Journal articles: 'Mycobacterium smegmatis'

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Chauhan, Priyanka, Santhe Amber van der Meulen, João Miguel Simões Caetano, Hojjat Ghasemi Goojani, Dennis Botman, Rob van Spanning, Holger Lill, and Dirk Bald. "Response of Mycobacterium smegmatis to the Cytochrome bcc Inhibitor Q203." International Journal of Molecular Sciences 23, no. 18 (September 7, 2022): 10331. http://dx.doi.org/10.3390/ijms231810331.

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For the design of next-generation tuberculosis chemotherapy, insight into bacterial defence against drugs is required. Currently, targeting respiration has attracted strong attention for combatting drug-resistant mycobacteria. Q203 (telacebec), an inhibitor of the cytochrome bcc complex in the mycobacterial respiratory chain, is currently evaluated in phase-2 clinical trials. Q203 has bacteriostatic activity against M. tuberculosis, which can be converted to bactericidal activity by concurrently inhibiting an alternative branch of the mycobacterial respiratory chain, cytochrome bd. In contrast, non-tuberculous mycobacteria, such as Mycobacterium smegmatis, show only very little sensitivity to Q203. In this report, we investigated factors that M. smegmatis employs to adapt to Q203 in the presence or absence of a functional cytochrome bd, especially regarding its terminal oxidases. In the presence of a functional cytochrome bd, M. smegmatis responds to Q203 by increasing the expression of cytochrome bcc as well as of cytochrome bd, whereas a M. smegmatisbd-KO strain adapted to Q203 by increasing the expression of cytochrome bcc. Interestingly, single-cell studies revealed cell-to-cell variability in drug adaptation. We also investigated the role of a putative second cytochrome bd isoform postulated for M. smegmatis. Although this putative isoform showed differential expression in response to Q203 in the M. smegmatisbd-KO strain, it did not display functional features similar to the characterised cytochrome bd variant.

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Converse, Scott E., and Jeffery S. Cox. "A Protein Secretion Pathway Critical for Mycobacterium tuberculosis Virulence Is Conserved and Functional in Mycobacterium smegmatis." Journal of Bacteriology 187, no. 4 (February 15, 2005): 1238–45. http://dx.doi.org/10.1128/jb.187.4.1238-1245.2005.

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ABSTRACT The Snm protein secretion system is a critical determinant of Mycobacterium tuberculosis virulence. However, genes encoding components of this pathway are conserved among all mycobacteria, including the nonpathogenic saprophyte Mycobacterium smegmatis. We show that the Snm system is operational in M. smegmatis and that secretion of its homologous ESAT-6 and CFP-10 substrates is regulated by growth conditions. Importantly, we show that Snm secretion in M. smegmatis requires genes that are homologous to those required for secretion in M. tuberculosis. Using a gene knockout strategy in M. smegmatis, we have also discovered four new gene products that are essential for Snm secretion, including the serine protease mycosin 1. Despite the evolutionary distance between M. smegmatis and M. tuberculosis, the M. smegmatis Snm system can secrete the M. tuberculosis ESAT-6 and CFP-10 proteins, suggesting that substrate recognition is also conserved between the two species. M. smegmatis, therefore, represents a powerful system to study the multicomponent Snm secretory machine and to understand the role of this conserved system in mycobacterial biology.

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Payton, Mark, Roy Auty, Rupika Delgoda, Martin Everett, and Edith Sim. "Cloning and Characterization of Arylamine N -Acetyltransferase Genes from Mycobacterium smegmatis and Mycobacterium tuberculosis: Increased Expression Results in Isoniazid Resistance." Journal of Bacteriology 181, no. 4 (February 15, 1999): 1343–47. http://dx.doi.org/10.1128/jb.181.4.1343-1347.1999.

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ABSTRACT Arylamine N-acetyltransferases (NATs) are found in many eukaryotic organisms, including humans, and have previously been identified in the prokaryote Salmonella typhimurium. NATs from many sources acetylate the antitubercular drug isoniazid and so inactivate it. nat genes were cloned fromMycobacterium smegmatis and Mycobacterium tuberculosis, and expressed in Escherichia coli andM. smegmatis. The induced M. smegmatis NAT catalyzes the acetylation of isoniazid. A monospecific antiserum raised against pure NAT from S. typhimurium recognizes NAT fromM. smegmatis and cross-reacts with recombinant NAT fromM. tuberculosis. Overexpression of mycobacterialnat genes in E. coli results in predominantly insoluble recombinant protein; however, with M. smegmatisas the host using the vector pACE-1, NAT proteins from M. tuberculosis and M. smegmatis are soluble. M. smegmatis transformants induced to express the M. tuberculosis nat gene in culture demonstrated a threefold higher resistance to isoniazid. We propose that NAT in mycobacteria could have a role in acetylating, and hence inactivating, isoniazid.

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Pandey, N., K. Singh, F. Ahmad, and R. Sharma. "Characterization of biofilm formation by Mycobacterium smegmatis during different environmental stress conditions: An in-vitro study." Journal of Environmental Biology 43, no. 6 (November 15, 2022): 771–78. http://dx.doi.org/10.22438/jeb/43/6/mrn-4081.

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Aim: In-vitro characterisation of biofilm produced by Mycobacterium smegmatis (M. smegmatis), a surrogate model for biofilm production by Mycobacteria, and to evaluate the impact of different environmental stress on mycobacterial growth and biofilm formation. Methodology: M. smegmatis biofilms were studied using tissue culture plate and tube adherence methods. Confocal Laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were used to study the 3D structure and surface morphology, respectively. Additionally, the effect of different environmental stress, such as the absence of essential ions, exposure to harsh environmental conditions, such as acidic environment or oxidative stress, on mycobacterial biofilm formation and mycobacterial growth was assessed. Results: All the exposures, except for carbon supplemented media had a detrimental effect on the number of viable counts and on biofilm formation by mycobacteria (p<0.001). Growth in low pH and oxidative stress was found to be maximum showing reduction by 98% when compared with control. Interpretation: Our findings present various environmental conditions that profoundly affect biofilm formation and thus, may find practical implications in future as effective mycobacterial control strategies having attributes of mycobacterial growth as well as biofilm inhibition. Key words: Biofilm, Environmental stress, Multi-drug resistance, Mycobacterium

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Guo, Ming, Zhonghe Sun, and Ying Zhang. "Mycobacterium smegmatis Has Two Pyrazinamidase Enzymes, PncA and PzaA." Journal of Bacteriology 182, no. 13 (July 1, 2000): 3881–84. http://dx.doi.org/10.1128/jb.182.13.3881-3884.2000.

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ABSTRACT The Mycobacterium smegmatis pncA gene, encoding nicotinamidase/pyrazinamidase, was identified. While it was similar to counterparts from other mycobacteria, the M. smegmatis PncA had little homology to the other M. smegmatispyrazinamidase/nicotinamidase, encoded by the pzaA gene. Transformation of Mycobacterium bovis strain BCG withM. smegmatis pncA or pzaA conferred susceptibility to pyrazinamide.

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Arthur, Patrick K., Vincent Amarh, Precious Cramer, Gloria B. Arkaifie, Ethel J. S. Blessie, Mohammed-Sherrif Fuseini, Isaac Carilo, Rebecca Yeboah, Leonard Asare, and Brian D. Robertson. "Characterization of Two New Multidrug-Resistant Strains of Mycobacterium smegmatis: Tools for Routine In Vitro Screening of Novel Anti-Mycobacterial Agents." Antibiotics 8, no. 1 (January 2, 2019): 4. http://dx.doi.org/10.3390/antibiotics8010004.

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Mycobacterium tuberculosis is a pathogen of global public health concern. This threat is exacerbated by the emergence of multidrug-resistant and extremely-drug-resistant strains of the pathogen. We have obtained two distinct clones of multidrug-resistant Mycobacterium smegmatis after gradual exposure of Mycobacterium smegmatis mc2 155 to increasing concentrations of erythromycin. The resulting resistant strains of Mycobacterium smegmatis exhibited robust viability in the presence of high concentrations of erythromycin and were found to be resistant to a wide range of other antimicrobials. They also displayed a unique growth phenotype in comparison to the parental drug-susceptible Mycobacterium smegmatis mc2 155, and a distinct colony morphology in the presence of cholesterol. We propose that these two multidrug-resistant clones of Mycobacterium smegmatis could be used as model organisms at the inceptive phase of routine in vitro screening of novel antimicrobial agents targeted against multidrug-resistant Mycobacterial tuberculosis.

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Burguière, Adeline, Paul G. Hitchen, Lynn G. Dover, Anne Dell, and Gurdyal S. Besra. "Altered expression profile of mycobacterial surface glycopeptidolipids following treatment with the antifungal azole inhibitors econazole and clotrimazole." Microbiology 151, no. 6 (June 1, 2005): 2087–95. http://dx.doi.org/10.1099/mic.0.27938-0.

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The azole antifungal drugs econazole and clotrimazole are known cytochrome P450 enzyme inhibitors. This study shows that these drugs are potent inhibitors of mycobacterial growth and are more effective against Mycobacterium smegmatis than isoniazid and ethionamide, two established anti-mycobacterial drugs. Several non-tuberculous mycobacteria, including the pathogenic members of the Mycobacterium avium–intracellulare complex (MAC) and the fast-growing saprophytic organism M. smegmatis, produce an array of serovar-specific (ss) and non-serovar-specific (ns) glycopeptidolipids (GPLs). GPL biosynthesis has been investigated for several years but has still not been fully elucidated. The authors demonstrate here that econazole and clotrimazole inhibit GPL biosynthesis in M. smegmatis. In particular, clotrimazole inhibits all four types of nsGPLs found in M. smegmatis, suggesting an early and common target within their biosynthetic pathway. Altogether, the data suggest that an azole-specific target, most likely a cytochrome P450, may be involved in the hydroxylation of the N-acyl chain in GPL biosynthesis. Azole antifungal drugs and potential derivatives could represent an interesting new range of anti-mycobacterial drugs, especially against opportunistic human pathogens including MAC, M. scrofulaceum, M. peregrinum, M. chelonae and M. abscessus.

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Cai, Xiaoying, Lei Liu, Chunhong Qiu, Chongzheng Wen, Yao He, Yanxiang Cui, Siyu Li, et al. "Identification and architecture of a putative secretion tube across mycobacterial outer envelope." Science Advances 7, no. 34 (August 2021): eabg5656. http://dx.doi.org/10.1126/sciadv.abg5656.

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Tuberculosis-causing mycobacteria have thick cell-wall and capsule layers that are formed from complex structures. Protein secretion across these barriers depends on a specialized protein secretion system, but none has been reported. We show that Mycobacterium tuberculosis Rv3705c and its homologous MSMEG_6251 in Mycobacterium smegmatis are tube-forming proteins in the mycobacterial envelope (TiME). Crystallographic and cryo-EM structures of these two proteins show that both proteins form rotationally symmetric rings. Two layers of TiME rings pack together in a tail-to-tail manner into a ring-shaped complex, which, in turn, stacks together to form tubes. M. smegmatis TiME was detected mainly in the cell wall and capsule. Knocking out the TiME gene markedly decreased the amount of secreted protein in the M. smegmatis culture medium, and expression of this gene in knocked-out strain partially restored the level of secreted protein. Our structure and functional data thus suggest that TiME forms a protein transport tube across the mycobacterial outer envelope.

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Grigorov, Artem S., Yulia V. Skvortsova, Oksana S. Bychenko, Leonid V. Aseev, Ludmila S. Koledinskaya, Irina V. Boni, and Tatyana L. Azhikina. "Dynamic Transcriptional Landscape of Mycobacterium smegmatis under Cold Stress." International Journal of Molecular Sciences 24, no. 16 (August 11, 2023): 12706. http://dx.doi.org/10.3390/ijms241612706.

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Bacterial adaptation to cold stress requires wide transcriptional reprogramming. However, the knowledge of molecular mechanisms underlying the cold stress response of mycobacteria is limited. We conducted comparative transcriptomic analysis of Mycobacterium smegmatis subjected to cold shock. The growth of M. smegmatis cultivated at 37oC was arrested just after exposure to cold (acclimation phase) but later (by 24 h) was resumed at a much slower rate (adaptation phase). Transcriptomic analyses revealed distinct gene expression patterns corresponding to the two phases. During the acclimation phase, differential expression was observed for genes associated with cell wall remodeling, starvation response, and osmotic pressure stress, in parallel with global changes in the expression of transcription factors and the downregulation of ribosomal genes, suggesting an energy-saving strategy to support survival. At the adaptation phase, the expression profiles were recovered, indicating restoration of the processes repressed earlier. Comparison of transcriptional responses in M. smegmatis with those in other bacteria revealed unique adaptation strategies developed by mycobacteria. Our findings shed light on the molecular mechanisms underlying M. smegmatis survival under cold stress. Further research should clarify whether the discovered transcriptional mechanisms exist in other mycobacterial species, including pathogenic Mycobacterium tuberculosis, which could be important for transmission control.

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Wolschendorf, Frank, Maysa Mahfoud, and Michael Niederweis. "Porins Are Required for Uptake of Phosphates by Mycobacterium smegmatis." Journal of Bacteriology 189, no. 6 (January 5, 2007): 2435–42. http://dx.doi.org/10.1128/jb.01600-06.

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ABSTRACT Phosphorus is an essential nutrient, but how phosphates cross the mycobacterial cell wall is unknown. Phosphatase activity in whole cells of Mycobacterium smegmatis was significantly lower than that in lysed cells, indicating that access to the substrate was restricted. The loss of the outer membrane (OM) porin MspA also reduced the phosphatase activity in whole cells compared to that in lysed cells. A similar result was obtained for M. smegmatis that overexpressed endogenous alkaline phosphatase, indicating that PhoA is not a surface protein, contrary to a previous report. The uptake of phosphate by a mutant lacking the porins MspA and MspC was twofold lower than that by wild-type M. smegmatis. Strikingly, the loss of these porins resulted in a severe growth defect of M. smegmatis on low-phosphate plates. We concluded that the OM of M. smegmatis represents a permeability barrier for phosphates and that Msp porins are the only OM channels for the diffusion of phosphate in M. smegmatis. However, phosphate diffusion through Msp pores is rather inefficient as shown by the 10-fold lower permeability of M. smegmatis for phosphate compared to that for glucose. This is likely due to the negative charges in the constriction zone of Msp porins. The phosphatase activity in whole cells of Mycobacterium bovis BCG was significantly less than that in lysed cells, indicating a similar uptake pathway for phosphates in slow-growing mycobacteria. However, porins that could mediate the diffusion of phosphates across the OM of M. bovis BCG and Mycobacterium tuberculosis are unknown.

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NIGOU, Jérôme, and Gurdyal S. BESRA. "Cytidine diphosphate-diacylglycerol synthesis in Mycobacterium smegmatis." Biochemical Journal 367, no. 1 (October 1, 2002): 157–62. http://dx.doi.org/10.1042/bj20020370.

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Recent studies have demonstrated that, during infection of macrophages by mycobacteria, phospholipids (PLs) are released from the mycobacterial cell wall within infected macrophages and transported out of this compartment into intracellular vesicles. The release of these PLs may have functions that influence the outcome of mycobacterial infections. Despite their important role, little is known about the biosynthesis of PLs in mycobacteria. In all organisms, PL biosynthesis begins with acylation of sn-glycerol 3-phosphate to form phosphatidic acid (PA), which is then converted to the central liponucleotide intermediate, cytidine diphosphate-diacylglycerol (CDP-DAG) via the CDP-DAG synthase (CDS). The present work examines CDS activity in Mycobacterium smegmatis extracts, with regard to subcellular localization, pH dependence, bivalent and univalent cation requirement, substrate specificity and regulation by nucleotides. We show that CDS activity, which is mainly found within the cytoplasmic membrane, is Mg2+-dependent and activated by K+ ions. Among PAs containing saturated fatty acids, dipalmitoyl-PA is the preferred substrate [Km = 0.23±0.03mM for Triton X-100 (v/v)/PA in the ratio 5:1]. Moreover, CDS activity is inhibited by the reaction products PPi (IC50 = 1.5mM), CDP-DAG (IC50 = 0.3mM) and the nucleotides ATP, UTP and GTP. This study contributes to the delineation of PL biosynthesis in mycobacteria.

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Dahl, John L., Kriti Arora, Helena I. Boshoff, Danelle C. Whiteford, Sophia A. Pacheco, Olaus J. Walsh, Dalia Lau-Bonilla, William B. Davis, and Anthony G. Garza. "The relA Homolog of Mycobacterium smegmatis Affects Cell Appearance, Viability, and Gene Expression." Journal of Bacteriology 187, no. 7 (April 1, 2005): 2439–47. http://dx.doi.org/10.1128/jb.187.7.2439-2447.2005.

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ABSTRACT The modification of metabolic pathways to allow for a dormant lifestyle appears to be an important feature for the survival of pathogenic bacteria within their host. One regulatory mechanism for persistent Mycobacterium tuberculosis infections is the stringent response. In this study, we analyze the stringent response of a nonpathogenic, saprophytic mycobacterial species, Mycobacterium smegmatis. The use of M. smegmatis as a tool for studying the mycobacterial stringent response was demonstrated by measuring the expression of two M. tuberculosis genes, hspX and eis, in M. smegmatis in the presence and absence of relMsm . The stringent response plays a role in M. smegmatis cellular and colony formation that is suggestive of changes in the bacterial cell wall structure.

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Poupin, P., J. J. Godon, E. Zumstein, and N. Truffaut. "Degradation of morpholine, piperidine, and pyrrolidine by mycobacteria: evidences for the involvement of a cytochrome P450." Canadian Journal of Microbiology 45, no. 3 (March 1, 1999): 209–16. http://dx.doi.org/10.1139/w99-002.

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Nine bacterial strains that grew on morpholine and pyrrolidine as sole carbon, nitrogen, and energy sources were isolated from three different environments with no known morpholine contamination. One of these strains could also degrade piperidine. These bacteria were identified as Mycobacterium strains. A phylogenetic analysis based on the partial 16S rDNA sequences indicated that the isolated strains clustered within the fast growing group of mycobacteria. When the above-mentioned cyclic amines were used as growth substrates, the synthesis of a soluble cytochrome P450 was induced in all these bacteria. Other laboratory strains, Mycobacterium fortuitum and Mycobacterium smegmatis mc2155, were tested for their abilities to degrade morpholine. Neither of them degraded morpholine but could use pyrrolidine and piperidine. The growth of M. fortuitum and M. smegmatis mc2155 on these compounds involved a soluble cytochrome P450, suggesting that mycobacterial strains are naturally able to use pyrrolidine and have developed a similar enzymatic pathway to metabolize this amine.Key words: mycobacteria, morpholine, piperidine, pyrrolidine, cytochrome P450.

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Korycka-Machala, Malgorzata, Ewelina Rychta, Anna Brzostek, Heather R. Sayer, Anna Rumijowska-Galewicz, Richard P. Bowater, and Jarosław Dziadek. "Evaluation of NAD+-Dependent DNA Ligase of Mycobacteria as a Potential Target for Antibiotics." Antimicrobial Agents and Chemotherapy 51, no. 8 (June 4, 2007): 2888–97. http://dx.doi.org/10.1128/aac.00254-07.

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ABSTRACT Mycobacteria contain genes for several DNA ligases, including ligA, which encodes a NAD+-dependent enzyme that has been postulated to be a target for novel antibacterial compounds. Using a homologous recombination system, direct evidence is presented that wild-type ligA cannot be deleted from the chromosome of Mycobacterium smegmatis. Deletions of native ligA in M. smegmatis could be obtained only after the integration of an extra copy of M. smegmatis or Mycobacterium tuberculosis ligA into the attB site of the chromosome, with expression controlled by chemically inducible promoters. The four ATP-dependent DNA ligases encoded by the M. smegmatis chromosome were unable to replace the function of LigA. Interestingly, the LigA protein from M. smegmatis could be substituted with the NAD+-dependent DNA ligase of Escherichia coli or the ATP-dependent ligase of bacteriophage T4. The conditional mutant strains allowed the analysis of the effect of LigA depletion on the growth of M. smegmatis. The protein level of the conditional mutants was estimated by Western blot analysis using antibodies raised against LigA of M. tuberculosis. This revealed that a strong overproduction or depletion of LigA did not affect the growth or survival of mycobacteria under standard laboratory conditions. In conclusion, although NAD+-dependent DNA ligase is essential for mycobacterial viability, only low levels of protein are required for growth. These findings suggest that very efficient inhibition of enzyme activity would be required if NAD+-dependent DNA ligase is to be useful as an antibiotic target in mycobacteria. The strains developed here will provide useful tools for the evaluation of the efficacy of any appropriate compounds in mycobacteria.

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Nguyen, Liem, Satheesh Chinnapapagari, and Charles J. Thompson. "FbpA-Dependent Biosynthesis of Trehalose Dimycolate Is Required for the Intrinsic Multidrug Resistance, Cell Wall Structure, and Colonial Morphology of Mycobacterium smegmatis." Journal of Bacteriology 187, no. 19 (October 1, 2005): 6603–11. http://dx.doi.org/10.1128/jb.187.19.6603-6611.2005.

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ABSTRACT Ligation of mycolic acids to structural components of the mycobacterial cell wall generates a hydrophobic, impermeable barrier that provides resistance to toxic compounds such as antibiotics. Secreted proteins FbpA, FbpB, and FbpC attach mycolic acids to arabinogalactan, generating mycolic acid methyl esters (MAME) or trehalose, generating α,α′-trehalose dimycolate (TDM; also called cord factor). Our studies of Mycobacterium smegmatis showed that disruption of fbpA did not affect MAME levels but resulted in a 45% reduction of TDM. The fbpA mutant displayed increased sensitivity to both front-line tuberculosis-targeted drugs as well as other broad-spectrum antibiotics widely used for antibacterial chemotherapy. The irregular, hydrophobic surface of wild-type M. smegmatis colonies became hydrophilic and smooth in the mutant. While expression of M. smegmatis fbpA restored defects of the mutant, heterologous expression of the Mycobacterium tuberculosis fbpA gene was less effective. A single mutation in the M. smegmatis FbpA esterase domain inactivated its ability to provide antibiotic resistance. These data show that production of TDM by FbpA is essential for the intrinsic antibiotic resistance and normal colonial morphology of some mycobacteria and support the concept that FbpA-specific inhibitors, alone or in combination with other antibiotics, could provide an effective treatment to tuberculosis and other mycobacterial diseases.

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Tran, Huyen Thi, Julia Solnier, Eva-Maria Pferschy-Wenzig, Olaf Kunert, Liam Martin, Sanjib Bhakta, Loi Huynh, Tri Minh Le, Rudolf Bauer, and Franz Bucar. "Antimicrobial and Efflux Pump Inhibitory Activity of Carvotacetones from Sphaeranthus africanus Against Mycobacteria." Antibiotics 9, no. 7 (July 8, 2020): 390. http://dx.doi.org/10.3390/antibiotics9070390.

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Carvotacetones (1–7) isolated from Sphaeranthus africanus were screened for their antimycobacterial and efflux pump (EP) inhibitory potential against the mycobacterial model strains Mycobacterium smegmatis mc2 155, Mycobacterium aurum ATCC 23366, and Mycobacterium bovis BCG ATCC 35734. The minimum inhibitory concentrations (MICs) of the carvotacetones were detected through high-throughput spot culture growth inhibition (HT-SPOTi) and microbroth dilution assays. In order to assess the potential of the compounds 1 and 6 to accumulate ethidium bromide (EtBr) in M. smegmatis and M. aurum, a microtiter plate-based fluorometric assay was used to determine efflux activity. Compounds 1 and 6 were analyzed for their modulating effects on the MIC of EtBr and the antibiotic rifampicin (RIF) against M. smegmatis. Carvotacetones 1 and 6 had potent antibacterial effects on M. aurum and M. bovis BCG (MIC ≤ 31.25 mg/L) and could successfully enhance EtBr activity against M. smegmatis. Compound 1 appeared as the most efficient agent for impairing the efflux mechanism in M. smegmatis. Both compounds 1 and 6 were highly effective against M. aurum and M. bovis BCG. In particular, compound 1 was identified as a valuable candidate for inhibiting mycobacterial efflux mechanisms and as a promising adjuvant in the therapy of tuberculosis or other non-tubercular mycobacterial infections.

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Titgemeyer, Fritz, Johannes Amon, Stephan Parche, Maysa Mahfoud, Johannes Bail, Maximilian Schlicht, Nadine Rehm, et al. "A Genomic View of Sugar Transport in Mycobacterium smegmatis and Mycobacterium tuberculosis." Journal of Bacteriology 189, no. 16 (June 8, 2007): 5903–15. http://dx.doi.org/10.1128/jb.00257-07.

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ABSTRACT We present a comprehensive analysis of carbohydrate uptake systems of the soil bacterium Mycobacterium smegmatis and the human pathogen Mycobacterium tuberculosis. Our results show that M. smegmatis has 28 putative carbohydrate transporters. The majority of sugar transport systems (19/28) in M. smegmatis belong to the ATP-binding cassette (ABC) transporter family. In contrast to previous reports, we identified genes encoding all components of the phosphotransferase system (PTS), including permeases for fructose, glucose, and dihydroxyacetone, in M. smegmatis. It is anticipated that the PTS of M. smegmatis plays an important role in the global control of carbon metabolism similar to those of other bacteria. M. smegmatis further possesses one putative glycerol facilitator of the major intrinsic protein family, four sugar permeases of the major facilitator superfamily, one of which was assigned as a glucose transporter, and one galactose permease of the sodium solute superfamily. Our predictions were validated by gene expression, growth, and sugar transport analyses. Strikingly, we detected only five sugar permeases in the slow-growing species M. tuberculosis, two of which occur in M. smegmatis. Genes for a PTS are missing in M. tuberculosis. Our analysis thus brings the diversity of carbohydrate uptake systems of fast- and a slow-growing mycobacteria to light, which reflects the lifestyles of M. smegmatis and M. tuberculosis in their natural habitats, the soil and the human body, respectively.

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Zimhony, Oren, Catherine Vilchèze, and William R. Jacobs. "Characterization of Mycobacterium smegmatis Expressing the Mycobacterium tuberculosis Fatty Acid Synthase I (fas1) Gene." Journal of Bacteriology 186, no. 13 (July 1, 2004): 4051–55. http://dx.doi.org/10.1128/jb.186.13.4051-4055.2004.

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ABSTRACT Unlike most other bacteria, mycobacteria make fatty acids with the multidomain enzyme eukaryote-like fatty acid synthase I (FASI). Previous studies have demonstrated that the tuberculosis drug pyrazinamide and 5-chloro-pyrazinamide target FASI activity. Biochemical studies have revealed that in addition to C16:0, Mycobacterium tuberculosis FASI synthesizes C26:0 fatty acid, while the Mycobacterium smegmatis enzyme makes C24:0 fatty acid. In order to express M. tuberculosis FASI in a rapidly growing Mycobacterium and to characterize the M. tuberculosis FASI in vivo, we constructed an M. smegmatis Δfas1 strain which contained the M. tuberculosis fas1 homologue. The M. smegmatis Δfas1 (attB::M. tuberculosis fas1) strain grew more slowly than the parental M. smegmatis strain and was more susceptible to 5-chloro-pyrazinamide. Surprisingly, while the M. smegmatis Δfas1 (attB::M. tuberculosis fas1) strain produced C26:0, it predominantly produced C24:0. These results suggest that the fatty acid elongation that produces C24:0 or C26:0 in vivo is due to a complex interaction among FASI, FabH, and FASII and possibly other systems and is not solely due to FASI elongation, as previously suggested by in vitro studies.

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Siqueira, Franciele Maboni, Cassiane Elizabete Lopes, Gustavo Geraldo Snell, and Marcos José Pereira Gomes. "Identification of Mycobacterium smegmatis in Bovine Mastitis." Acta Scientiae Veterinariae 44, no. 1 (January 16, 2016): 4. http://dx.doi.org/10.22456/1679-9216.83088.

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Background: Rapidly growing mycobacteria (RGM) are ubiquitous in the environment, can be isolated from soil and water, and demonstrate visible growth on culture media within seven days. Mycobacterium smegmatis is an acid-alcohol fast bacterium, which belong to RGM group. The diagnosis of M. smegmatis infections may be quite difficult by conventional methods; therefore, biochemistry associated to nucleic acid-based approaches provided fast and accurate identification. Although this specie may be associated to animals and humans infections, there is few cases description. Nontuberculous mycobacterial bovine mastitis is uncommon, and bovine mastitis by M. smegmatis has been reported but non-confirmed case once in the past. This paper reports M. smegmatis recovered from a cattle with relapsing pyogranulomatous mastitis.Case: Milk samples from an adult Holstein cow showing relapsing pyogranulomatous mastitis history and by pronounced glandular hardening were cultivated and analyzed accordingly to standard milk cultivation protocols. The animal had been subject to several intramammary and parenteral antibiotic therapies protocols without adequate response. After 48 h incubation, a slow and sparse growth of slightly pigmented, shiny and smooth colonies was observed on the blood agar plate. The bacterium isolated was named as strain 55/08. The morphological and biochemical profile were tested, and the ability of the isolate to grow at Lowenstein-Jensen slants was confirmed. The isolated have showed positive reaction to catalase, glucose, sucrose, mannitol and nitrate. The pigment formation was observed for 14 days incubation, and the colonies produce pigment after prolonged time. Gram and Ziehl-Neelsen staining revealed poorly pigmented, irregular, slender Gram-positive and acid fast rods. The staining and biochemical profile showed closed isolated relationship to M. smegmatis. A discriminatory identification based in the 16S rRNA gene sequence analysis was performed. The total DNA from the strain 55/08 was extracted and the partial 16S rRNA sequence was amplified, using prokaryotic universal primer pairs and the extract DNA as template, by PCR assay following the purification and sequencing of the amplicons. A total of 1,443 nucleotides form consensus sequence were alignment to M. smegmatis and other mycobacteria 16S rRNA available sequences. The sequence analysis confirmed the M. smegmatis identification as etiological agent of bovine relapsing pyogranulomatous mastitis. M. smegmatis strain 55/08 partial 16S rRNA gene sequence was submitted to GenBank. The phylogenetic relationship of the strain 55/08 with other mycobacteria was performed in order to confirm the identification of the isolate as M. smegmatis. Discussion: Nontuberculous mycobacteria are uncommon causes of bovine mastitis. Some old reports have described M. smegmatis as etiological agent of mastitis, but without definitive diagnostic. M. smegmatis mammary quarter introduction may be related to the repeated intramammary treatment protocols, because this mycobacteria is related to environmental infections. The relapsing pyogranulomatous mastitis infection could be associated to other bacteria species. However, the phenotypic and molecular characterization which was performed demonstrated the accurate identification of the isolated as M. smegmatis. Milk contaminated by M. smegmatis may be a potential infection source for human and other animal species. This report reinforces the need to optimize quality programs and laboratorial diagnosis to further the accurate microorganism identification in milk samples.Keywords: RGM mycobacteria, relapsing mastitis, M. smegmatis, molecular identification.

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Elamin, Oumaima, Marwa Chraibi, Saad Koraichi Ibnsouda, and Mohammed Houssaini Iraqui. "LYCOPENE PRODUCTION IN MYCOBACTERIUM SMEGMATIS BY EXPRESSION OF CRT GENES FROM MYCOBACTERIUM AURUM AND PROTECTIVE EFFECT OF LYCOPENE IN VIVO AND IN VITRO AGAINST UV RADIATION." International Journal of Pharmacy and Pharmaceutical Sciences 10, no. 9 (September 1, 2018): 49. http://dx.doi.org/10.22159/ijpps.2018v10i9.25768.

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Objective: The aim of the present study was to express in Mycobacterium smegmatis the clustered mycobacterial genes coding for lycopene synthesis and to investigate the protective properties of lycopene against ultraviolet (UV) irradiation.Methods: The genes, which encode the biogenesis of lycopene in Mycobacterium aurum A+, were introduced into Mycobacterium smegmatis by electroporation. The pigments produced were analyzed by thin layer chromatography, and the absorption spectra were determined. A survival test using UV irradiations was also performed.Results: The transformed Mycobacterium smegmatis were found to synthesize lycopene with important yield (1.41± 3.09 mg/g) and was more resistant to ultraviolet irradiation than non-pigmented strain (p<0.01). Furthermore, cells of M. smegmatis not transformed but coated with lycopene are more resistant to UV than those uncoated (p<0.01).Conclusion: M. smegmatis can form orange colonies on agar plates when it is transformed with the lycopene genes, and the transformants produces 1.41 mg/g (dry weight) of this carotene. Our findings strongly suggest that lycopene has antioxidant activities and prevent the lethal action of UV irradiation on bacterial cells in vivo and in vitro, and deserves further studies considering the amelioration of the production.

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Jeevarajah, Dharshini, John H. Patterson, Ellen Taig, Tobias Sargeant, Malcolm J. McConville, and Helen Billman-Jacobe. "Methylation of GPLs in Mycobacterium smegmatis and Mycobacterium avium." Journal of Bacteriology 186, no. 20 (October 15, 2004): 6792–99. http://dx.doi.org/10.1128/jb.186.20.6792-6799.2004.

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ABSTRACT Several species of mycobacteria express abundant glycopeptidolipids (GPLs) on the surfaces of their cells. The GPLs are glycolipids that contain modified sugars including acetylated 6-deoxy-talose and methylated rhamnose. Four methyltransferases have been implicated in the synthesis of the GPLs of Mycobacterium smegmatis and Mycobacterium avium. A rhamnosyl 3-O-methytransferase and a fatty acid methyltransferase of M. smegmatis have been previously characterized. In this paper, we characterize the methyltransferases that are responsible for modifying the hydroxyl groups at positions 2 and 4 of rhamnose and propose the biosynthetic sequence of GPL trimethylrhamnose formation. The analysis of M. avium genes through the creation of specific mutants is technically difficult; therefore, an alternative approach to determine the function of putative methyltransferases of M. avium was undertaken. Complementation of M. smegmatis methyltransferase mutants with M. avium genes revealed that MtfC and MtfB of the latter species have 4-O-methyltransferase activity and that MtfD is a 3-O-methyltransferase which can modify rhamnose of GPLs in M. smegmatis.

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Sharbati-Tehrani, Soroush, Joachim Stephan, Gudrun Holland, Bernd Appel, Michael Niederweis, and Astrid Lewin. "Porins limit the intracellular persistence of Mycobacterium smegmatis." Microbiology 151, no. 7 (July 1, 2005): 2403–10. http://dx.doi.org/10.1099/mic.0.27969-0.

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The genus Mycobacterium comprises highly pathogenic as well as opportunistic or apathogenic species exhibiting a great variability with respect to their ability to persist or multiply within monocytic host cells. The impact of the permeability of the mycobacterial outer membrane on intracellular persistence was studied. For this purpose, a Mycobacterium smegmatis mutant with a deletion of the major porin gene mspA and a second mutant lacking mspA and the homologous porin gene mspC were used. Deletion of mspA together with mspC significantly enhanced intracellular persistence in murine bone marrow macrophages, the mouse macrophage cell line J774A.1 and Acanthamoeba castellanii. Complementation of mspA in the porin mutant strains resulted in restoration of the wild-type phenotype with respect to intracellular persistence. This is the first report to show that the deletion of porins of mycobacteria results in improved persistence in eukaryotic cells, demonstrating that the intracellular persistence of M. smegmatis depends upon the permeability of the outer membrane.

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Dziadek, Jaroslaw, Stacey A. Rutherford, Murty V. Madiraju, Mark A. L. Atkinson, and Malini Rajagopalan. "Conditional expression of Mycobacterium smegmatis ftsZ, an essential cell division gene." Microbiology 149, no. 6 (June 1, 2003): 1593–603. http://dx.doi.org/10.1099/mic.0.26023-0.

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To understand the role of Mycobacterium smegmatis ftsZ (ftsZsmeg ) in the cell division process, the ftsZ gene was characterized at the genetic level. This study shows that ftsZsmeg is an essential gene in that it can only be disrupted in a merodiploid background carrying another functional copy. Expression of ftsZsmeg in M. smegmatis from a constitutively active mycobacterial promoter resulted in lethality whereas that from a chemically inducible acetamidase (ami) promoter led to FtsZ accumulation, filamentation and cell lysis. To further understand the roles of ftsZ in cell division a conditionally complementing ftsZsmeg mutant strain was constructed in which ftsZ expression is controlled by acetamide. Growth in the presence of 0·2 % acetamide increased FtsZ levels approximately 1·4-fold, but did not decrease viability or change cell length. Withdrawal of acetamide reduced FtsZ levels, decreased viability, increased cell length and eventually lysed the cells. Finally, it is shown that ftsZsmeg function in M. smegmatis can be replaced with the Mycobacterium tuberculosis counterpart, indicating that heterologous FtsZ tb can independently initiate the formation of Z-rings and catalyse the septation process. It is concluded that optimal levels of M. smegmatis FtsZ are required to sustain cell division and that the cell division initiation mechanisms are similar in mycobacteria.

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Udou, Takezo. "Extracellular hemolytic activity in rapidly growing mycobacteria." Canadian Journal of Microbiology 40, no. 4 (April 1, 1994): 318–21. http://dx.doi.org/10.1139/m94-052.

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Little is known about virulence factors associated with rapidly growing mycobacteria. We evaluated 42 clinical isolates of Mycobacterium fortuitum and Mycobacterium chelonae and 4 reference strains of Mycobacterium smegmatis for the production of hemolysin (or hemolytic substance) as a possible contributor to the pathogenesis of disease caused by these organisms. All the strains tested possessed extracellular hemolytic activity that was stable after heating and proteinase treatment, and the active substance had a molecular weight less than 10 000. The activity accumulated in culture medium during the late exponential to mid stationary phase of growth. Hemolysis in vitro was relatively slow; incubation for 10 h at 35 °C was required to obtain maximal activity. Some specificity of the hemolysis with regard to red blood cells from different animals was observed.Key words: rapidly growing mycobacteria, Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium smegmatis, hemolytic activity.

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Nash, Kevin A. "Intrinsic Macrolide Resistance in Mycobacterium smegmatis Is Conferred by a Novel erm Gene, erm(38)." Antimicrobial Agents and Chemotherapy 47, no. 10 (October 2003): 3053–60. http://dx.doi.org/10.1128/aac.47.10.3053-3060.2003.

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ABSTRACT High-level, acquired macrolide resistance in mycobacteria is conferred by mutation within the 23S rRNA gene. However, several mycobacteria are naturally resistant to macrolides, including the Mycobacterium smegmatis group and Mycobacterium tuberculosis complex. Thus, the aim of this study was to characterize this resistance. Intrinsic macrolide resistance in M. smegmatis was inducible and showed cross-resistance to lincosamides but not to streptogramin B (i.e., ML resistance). A similar phenotype was found with Mycobacterium microti and macrolide-resistant Mycobacterium fortuitum. A search of the DNA sequence data for M. smegmatis strain mc2155 identified a novel erm gene, erm(38), and expression analysis showed that erm(38) RNA levels increased >10-fold after a 2-h incubation with macrolide. Inducible ML resistance was not expressed by an erm(38) knockout mutant, and complementation of this mutant with intact erm(38) in trans resulted in high-level ML resistance (e.g., clarithromycin MIC of >512 μg/ml). Thus, the results indicate that erm(38) confers the intrinsic ML resistance of M. smegmatis. Southern blot analysis with an erm(38)-specific probe indicated that a similar gene may be present in macrolide-resistant M. fortuitum. This finding, with the presence of the erm(37) gene (Rv1988) in the M. tuberculosis complex, suggests that such genes are widespread in mycobacteria with intrinsic macrolide resistance.

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Mo, Yongkai, Natalie M. Quanquin, William H. Vecino, Uma Devi Ranganathan, Lydia Tesfa, William Bourn, Keith M. Derbyshire, Norman L. Letvin, William R. Jacobs, and Glenn J. Fennelly. "Genetic Alteration of Mycobacterium smegmatis To Improve Mycobacterium-Mediated Transfer of Plasmid DNA into Mammalian Cells and DNA Immunization." Infection and Immunity 75, no. 10 (July 30, 2007): 4804–16. http://dx.doi.org/10.1128/iai.01877-06.

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ABSTRACT Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and fluorescence-activated cell sorting analysis after infection of phagocytic and nonphagocytic cell lines by Mycobacterium smegmatis or M. bovis BCG harboring a plasmid encoding the fluorescence gene under the control of a eukaryotic promoter. Next, we compared the efficiencies of gene transfer using M. smegmatis or BCG containing chromosomal insertions or deletions that cause early lysis, hyperconjugation, or an increased plasmid copy number. We observed a significant—albeit only 1.7-fold—increase in the level of plasmid transfer to eukaryotic cells infected with M. smegmatis hyperconjugation mutants. M. smegmatis strains that overexpressed replication proteins (Rep) of pAL5000, a plasmid whose replicon is incorporated in many mycobacterial constructs, generated a 10-fold increase in plasmid copy number and 3.5-fold and 3-fold increases in gene transfer efficiency to HeLa cells and J774 cells, respectively. Although BCG strains overexpressing Rep could not be recovered, BCG harboring a plasmid with a copy-up mutation in oriM resulted in a threefold increase in gene transfer to J774 cells. Moreover, M. smegmatis strains overexpressing Rep enhanced gene transfer in vivo compared with a wild-type control. Immunization of mice with mycobacteria harboring a plasmid (pgp120h E) encoding human immunodeficiency virus gp120 elicited gp120-specific CD8 T-cell responses among splenocytes and peripheral blood mononuclear cells that were up to twofold (P < 0.05) and threefold (P < 0.001) higher, respectively, in strains supporting higher copy numbers. The magnitude of these responses was approximately one-half of that observed after intramuscular immunization with pgp120h E. M. smegmatis and other nonpathogenic mycobacteria are promising candidate vectors for DNA vaccine delivery.

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ERGAN, Begum, Lutfi COPLU, Alpaslan ALP, and Mustafa ARTVINLI. "Mycobacterium smegmatis pneumonia." Respirology 9, no. 2 (June 2004): 283–85. http://dx.doi.org/10.1111/j.1440-1843.2004.00570.x.

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Gibbons, Henry S., Frank Wolschendorf, Michelle Abshire, Michael Niederweis, and Miriam Braunstein. "Identification of Two Mycobacterium smegmatis Lipoproteins Exported by a SecA2-Dependent Pathway." Journal of Bacteriology 189, no. 14 (May 11, 2007): 5090–100. http://dx.doi.org/10.1128/jb.00163-07.

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ABSTRACT The SecA2 protein is part of a specialized protein export system of mycobacteria. We set out to identify proteins exported to the bacterial cell envelope by the mycobacterial SecA2 system. By comparing the protein profiles of cell wall and membrane fractions from wild-type and ΔsecA2 mutant Mycobacterium smegmatis, we identified the Msmeg1712 and Msmeg1704 proteins as SecA2-dependent cell envelope proteins. These are the first endogenous M. smegmatis proteins identified as dependent on SecA2 for export. Both proteins are homologous to periplasmic sugar-binding proteins of other bacteria, and both contain functional amino-terminal signal sequences with lipobox motifs. These two proteins appeared to be genuine lipoproteins as shown by Triton X-114 fractionation and sensitivity to globomycin, an inhibitor of lipoprotein signal peptidase. The role of SecA2 in the export of these proteins was specific; not all mycobacterial lipoproteins required SecA2 for efficient localization or processing. Finally, Msmeg1704 was recognized by the SecA2 pathway of Mycobacterium tuberculosis, as indicated by the appearance of an export intermediate when the protein was expressed in a ΔsecA2 mutant of M. tuberculosis. Taken together, these results indicate that a select subset of envelope proteins containing amino-terminal signal sequences can be substrates of the mycobacterial SecA2 pathway and that some determinants for SecA2-dependent export are conserved between M. smegmatis and M. tuberculosis.

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Cougoule, Céline, Patricia Constant, Gilles Etienne, Mamadou Daffé, and Isabelle Maridonneau-Parini. "Lack of Fusion of Azurophil Granules with Phagosomes during Phagocytosis of Mycobacterium smegmatis by Human Neutrophils Is Not Actively Controlled by the Bacterium." Infection and Immunity 70, no. 3 (March 2002): 1591–98. http://dx.doi.org/10.1128/iai.70.3.1591-1598.2002.

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ABSTRACT Biogenesis of phagolysosomes is a very rapid event in neutrophils which takes place with nascent unclosed phagosomes, leading to the release of lysosomal enzymes such as β-glucuronidase in the extracellular medium. We have previously shown that, under nonopsonic conditions, both pathogenic and nonpathogenic mycobacteria uncouple phagocytosis from fusion of azurophil granules (specialized secretory lysosomes) with phagosomes. In the present study we questioned whether they actively act on neutrophils to block this process or use phagocytic receptors that negatively control the biogenesis of phagolysosomes. As for live unicellular Mycobacterium smegmatis, we observed that nonopsonic phagocytosis of heat-killed mycobacteria did not induce the release of β-glucuronidase, indicating that M. smegmatis does not actively act on the fusion process in neutrophils. In contrast, phagocytosis of unicellular M. smegmatis opsonized in immune serum or that of small nonopsonized mycobacterial aggregates restored the biogenesis of phagolysosomes. Aggregates were internalized in a CR3- and cholesterol-dependent manner as unicellular mycobacteria. However, aggregates but not unicellular bacteria triggered F-actin and Hck recruitment at the phagosomes, events that have been associated with lysosome fusion. Thus, we propose that M. smegmatis does not actively control the fusion of azurophil granules at early time points postinfection and that mycobacterial aggregates recruit large clusters of receptors at the neutrophil surface which could trap proteins implicated in the biogenesis of phagolysosomes.

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El-Etr, Sahar H., Ling Yan, and Jeffrey D. Cirillo. "Fish Monocytes as a Model for Mycobacterial Host-Pathogen Interactions." Infection and Immunity 69, no. 12 (December 1, 2001): 7310–17. http://dx.doi.org/10.1128/iai.69.12.7310-7317.2001.

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ABSTRACT Mycobacterium marinum, a relatively rapid-growing fish and human pathogen, has become an important model for the investigation of mycobacterial pathogenesis. M. marinumis closely related to the Mycobacterium tuberculosiscomplex and causes a disease in fish and amphibians with pathology similar to tuberculosis. We have developed an in vitro model for the study of M. marinum virulence mechanisms using the carp monocytic cell line CLC (carp leukocyte culture). We found that fish monocytes can differentiate between pathogenic and nonpathogenic mycobacterial species. Interestingly, M. marinum enters fish monocytes at a 40- to 60-fold-higher rate thanMycobacterium smegmatis. In addition, M. marinum survives and replicates in fish monocytes whileM. smegmatis is killed. We also found that M. marinum inhibits lysosomal fusion in fish monocytes, indicating that these cells may be used to dissect the mechanisms of intracellular trafficking in mycobacteria. We conclude from these observations that monocytic cells from fish, a natural host for M. marinum, provide an extremely valuable model for the identification and characterization of mycobacterial virulence determinants in the laboratory.

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Do, Thi Thuy, Jerónimo Rodríguez-Beltran, Esmeralda Cebrián-Sastre, Alexandro Rodríguez-Rojas, Alfredo Castañeda-García, and Jesús Blázquez. "Inactivation of a New Potassium Channel Increases Rifampicin Resistance and Induces Collateral Sensitivity to Hydrophilic Antibiotics in Mycobacterium smegmatis." Antibiotics 11, no. 4 (April 12, 2022): 509. http://dx.doi.org/10.3390/antibiotics11040509.

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Rifampicin is a critical first-line antibiotic for treating mycobacterial infections such as tuberculosis, one of the most serious infectious diseases worldwide. Rifampicin resistance in mycobacteria is mainly caused by mutations in the rpoB gene; however, some rifampicin-resistant strains showed no rpoB mutations. Therefore, alternative mechanisms must explain this resistance in mycobacteria. In this work, a library of 11,000 Mycobacterium smegmatis mc2 155 insertion mutants was explored to search and characterize new rifampicin-resistance determinants. A transposon insertion in the MSMEG_1945 gene modified the growth rate, pH homeostasis and membrane potential in M. smegmatis, producing rifampicin resistance and collateral susceptibility to other antitubercular drugs such as isoniazid, ethionamide and aminoglycosides. Our data suggest that the M. smegmatis MSMEG_1945 protein is an ion channel, dubbed MchK, essential for maintaining the cellular ionic balance and membrane potential, modulating susceptibility to antimycobacterial agents. The functions of this new gene point once again to potassium homeostasis impairment as a proxy to resistance to rifampicin. This study increases the known repertoire of mycobacterial ion channels involved in drug susceptibility/resistance to antimycobacterial drugs and suggests novel intervention opportunities, highlighting ion channels as druggable pathways.

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Raghunand, Tirumalai R., and William R. Bishai. "Mycobacterium smegmatis whmD and its homologue Mycobacterium tuberculosis whiB2 are functionally equivalent." Microbiology 152, no. 9 (September 1, 2006): 2735–47. http://dx.doi.org/10.1099/mic.0.28911-0.

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Mycobacterium smegmatis whmD is is an essential gene involved in cell division. This paper shows that whmD and its homologue whiB2 in Mycobacterium tuberculosis are functionally equivalent. The genes are syntenous, and share significant homology in both their coding and non-coding DNA sequences. Transcription site mapping showed that the two genes possess near-identical promoter elements, and they displayed comparable promoter strengths in a reporter gene assay. The two proteins show near identity in their C-terminus, and polyclonal antiserum to WhmD specifically cross-reacts with a ∼15 kDa band in M. tuberculosis lysates. Following overexpression of sense and anti-sense constructs in their cognate mycobacterial hosts, whiB2 and whmD transformants displayed a small-colony phenotype, exhibited filamentation, and showed a reduction in viability. These observations reveal that the two proteins are functionally homologous and that their intracellular concentration is critical for septation in mycobacteria. Colonies of M. tuberculosis overexpressing whiB2 were spherical and glossy, suggesting a change in composition of the cell envelope. Filaments of the conditionally complemented M. smegmatis whmD mutant were non-acid-fast, also indicating changes in characteristics of surface lipids. M. smegmatis transformants carrying a whmD–gfp fusion showed a diffuse pattern of fluorescence, consistent with the putative role of WhmD as a regulator. These observations strongly suggest that M. tuberculosis whiB2 is an essential gene and its protein product in all likelihood regulates the expression of genes involved in the cell division cascade.

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Radhakrishnan, Anjana, Christopher M. Furze, Mohd Syed Ahangar, and Elizabeth Fullam. "A GFP-strategy for efficient recombinant protein overexpression and purification in Mycobacterium smegmatis." RSC Advances 8, no. 58 (2018): 33087–95. http://dx.doi.org/10.1039/c8ra06237d.

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Abdalla, Abualgasim Elgaili, Shuangquan Yan, Jie Zeng, Wanyan Deng, Longxiang Xie, and Jianping Xie. "Mycobacterium tuberculosis Rv0341 Promotes Mycobacterium Survival in In Vitro Hostile Environments and within Macrophages and Induces Cytokines Expression." Pathogens 9, no. 6 (June 8, 2020): 454. http://dx.doi.org/10.3390/pathogens9060454.

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Mycobacterium tuberculosis represents an ancient deadly human pathogen that can survive and multiply within macrophages. The effectors are key players for the successful pathogenesis of this bacterium. M. tuberculosis open reading frame (ORF) Rv0341, a pathogenic mycobacteria-specific gene, was found to be upregulated in macrophages isolated from human tuberculosis granuloma and inside the macrophages during in vitro infection by M. tuberculosis. To understand the exact role of this gene, we expressed the Rv0341 gene in M. smegmatis, which is a non-pathogenic Mycobacterium. We found that Rv0341 expression can alter colony morphology, reduce the sliding capability, and decrease the cell wall permeability of M. smegmatis. Furthermore, Rv0341 remarkably enhanced M. smegmatis survival within macrophages and under multiple in vitro stress conditions when compared with the control strain. Ms_Rv0341 significantly induced expression of TNF-α, IL-1β, and IL-10 compared with M. smegmatis harboring an empty vector. In summary, these data suggest that Rv0341 is one of the M. tuberculosis virulence determinants that can promote bacilli survival in harsh conditions and inside macrophages.

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Ezquerra-Aznárez, José Manuel, Giulia Degiacomi, Henrich Gašparovič, Giovanni Stelitano, Josè Camilla Sammartino, Jana Korduláková, Paolo Governa, et al. "The Veterinary Anti-Parasitic Selamectin Is a Novel Inhibitor of the Mycobacterium tuberculosis DprE1 Enzyme." International Journal of Molecular Sciences 23, no. 2 (January 11, 2022): 771. http://dx.doi.org/10.3390/ijms23020771.

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Avermectins are macrocyclic lactones with anthelmintic activity. Recently, they were found to be effective against Mycobacterium tuberculosis, which accounts for one third of the worldwide deaths from antimicrobial resistance. However, their anti-mycobacterial mode of action remains to be elucidated. The activity of selamectin was determined against a panel of M. tuberculosis mutants. Two strains carrying mutations in DprE1, the decaprenylphosphoryl-β-D-ribose oxidase involved in the synthesis of mycobacterial arabinogalactan, were more susceptible to selamectin. Biochemical assays against the Mycobacterium smegmatis DprE1 protein confirmed this finding, and docking studies predicted a binding site in a loop that included Leu275. Sequence alignment revealed variants in this position among mycobacterial species, with the size and hydrophobicity of the residue correlating with their MIC values; M. smegmatis DprE1 variants carrying these point mutations validated the docking predictions. However, the correlation was not confirmed when M. smegmatis mutant strains were constructed and MIC phenotypic assays performed. Likewise, metabolic labeling of selamectin-treated M. smegmatis and M. tuberculosis cells with 14C-labeled acetate did not reveal the expected lipid profile associated with DprE1 inhibition. Together, our results confirm the in vitro interactions of selamectin and DprE1 but suggest that selamectin could be a multi-target anti-mycobacterial compound.

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Su, Chih-Chia, Philip A. Klenotic, Meng Cui, Meinan Lyu, Christopher E. Morgan, and Edward W. Yu. "Structures of the mycobacterial membrane protein MmpL3 reveal its mechanism of lipid transport." PLOS Biology 19, no. 8 (August 12, 2021): e3001370. http://dx.doi.org/10.1371/journal.pbio.3001370.

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The mycobacterial membrane protein large 3 (MmpL3) transporter is essential and required for shuttling the lipid trehalose monomycolate (TMM), a precursor of mycolic acid (MA)-containing trehalose dimycolate (TDM) and mycolyl arabinogalactan peptidoglycan (mAGP), in Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium smegmatis. However, the mechanism that MmpL3 uses to facilitate the transport of fatty acids and lipidic elements to the mycobacterial cell wall remains elusive. Here, we report 7 structures of the M. smegmatis MmpL3 transporter in its unbound state and in complex with trehalose 6-decanoate (T6D) or TMM using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography. Combined with calculated results from molecular dynamics (MD) and target MD simulations, we reveal a lipid transport mechanism that involves a coupled movement of the periplasmic domain and transmembrane helices of the MmpL3 transporter that facilitates the shuttling of lipids to the mycobacterial cell wall.

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Liu, Hanrui, Xuwen Gui, Shixing Chen, Weizhe Fu, Xiang Li, Tingyuan Xiao, Jie Hou, and Tao Jiang. "Structural Variability of Lipoarabinomannan Modulates Innate Immune Responses within Infected Alveolar Epithelial Cells." Cells 11, no. 3 (January 21, 2022): 361. http://dx.doi.org/10.3390/cells11030361.

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Mycobacterium tuberculosis (M. tb) is an intracellular pathogen persisting in phagosomes that has the ability to escape host immune surveillance causing tuberculosis (TB). Lipoarabinomannan (LAM), as a glycolipid, is one of the complex outermost components of the mycobacterial cell envelope and plays a critical role in modulating host responses during M. tb infection. Different species within the Mycobacterium genus exhibit distinct LAM structures and elicit diverse innate immune responses. However, little is known about the mechanisms. In this study, we first constructed a LAM-truncated mutant with fewer arabinofuranose (Araf) residues named M. sm-ΔM_6387 (Mycobacterium smegmatis arabinosyltransferase EmbC gene knockout strain). It exhibited some prominent cell wall defects, including tardiness of mycobacterial migration, loss of acid-fast staining, and increased cell wall permeability. Within alveolar epithelial cells (A549) infected by M. sm-ΔM_6387, the uptake rate was lower, phagosomes with bacterial degradation appeared, and microtubule-associated protein light chain 3 (LC3) recruitment was enhanced compared to wild type Mycobacterium smegmatis (M. smegmatis). We further confirmed that the variability in the removal capability of M. sm-ΔM_6387 resulted from host cell responses rather than the changes in the mycobacterial cell envelope. Moreover, we found that M. sm-ΔM_6387 or its glycolipid extracts significantly induced expression changes in some genes related to innate immune responses, including Toll-like receptor 2 (TLR2), class A scavenger receptor (SR-A), Rubicon, LC3, tumor necrosis factor alpha (TNF-α), Bcl-2, and Bax. Therefore, our studies suggest that nonpathogenic M. smegmatis can deposit LC3 on phagosomal membranes, and the decrease in the quantity of Araf residues for LAM molecules not only impacts mycobacterial cell wall integrity but also enhances host defense responses against the intracellular pathogens and decreases phagocytosis of host cells.

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Šimunović, Katarina, Julia Solnier, Fabian Alperth, Olaf Kunert, Sonja Smole Smole Možina, and Franz Bucar. "Efflux Pump Inhibition and Resistance Modulation in Mycobacterium smegmatis by Peucedanum ostruthium and Its Coumarins." Antibiotics 10, no. 9 (September 5, 2021): 1075. http://dx.doi.org/10.3390/antibiotics10091075.

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Antibiotic resistance is a growing problem and may become the next major global health crisis if no timely actions are taken. Mycobacterial infections are widespread and, due to antibiotic resistance, also hard to treat and a major cause of mortality. Natural compounds have the potential to increase antibiotic effectiveness due to their resistance modulatory and antimicrobial effects. In this study, Peucedanum ostruthium extracts, fractions, and isolated compounds were investigated regarding their antimicrobial and resistance-modulatory effects as well as efflux pump inhibition in Mycobacterium smegmatis. P. ostruthium extracts were found to have anti-mycobacterial potential and resistance modulating effects on ethidium bromide activity. The major antibacterial effect was attributed to ostruthin, and we found that the more lipophilic the substrate, the greater the antimicrobial effect. Imperatorin caused potent modulatory effects by interfering with the action of the major LfrA efflux pump in M. smegmatis. The plant P. ostruthuim has a complex effect on M. smegmatis, including antibacterial, efflux pump inhibition, resistance modulation, and membrane permeabilization, and its major constituents, ostruthin and imperatorin, have a distinct role in these effects. This makes P. ostruthium and its coumarins promising therapeutics to consider in the fight against drug-resistant mycobacteria.

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39

Cascioferro, Alessandro, Francesca Boldrin, Agnese Serafini, Roberta Provvedi, Giorgio PalÃ¯Â¿Â½, and Riccardo Manganelli. "Xer Site-Specific Recombination, an Efficient Tool To Introduce Unmarked Deletions into Mycobacteria." Applied and Environmental Microbiology 76, no. 15 (June 11, 2010): 5312–16. http://dx.doi.org/10.1128/aem.00382-10.

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ABSTRACT Genetic manipulation of mycobacteria still represents a serious challenge due to the lack of tools and selection markers. In this report, we describe the development of an intrinsically unstable excisable cassette for introduction of unmarked mutations in both Mycobacterium smegmatis and Mycobacterium tuberculosis.

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Lima, Andrea Santos, Maria Madileuza Carneiro Neves, Karen Machado Gomes, Klarissa Miranda Guarines, Carlos Feitosa Luna, Rafael Silva Duarte, Lílian Maria Lapa Montenegro, and Haiana Charifker Schindler. "First case report of infection by Mycobacterium wolinskyi after mammoplasty in Brazil." Infectious Disease Reports 5, no. 2 (October 14, 2013): 12. http://dx.doi.org/10.4081/idr.2013.e12.

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Mycobacterium wolinskyi is a rapidly growing mycobacterium, first described in 1999 as a member of the group Mycobacterium smegmatis (Mycobacterium smegmatis, Mycobacterium wolinskyi and Mycobacterium goodii). Only 19 case reports all over the world have been described on literature, none of them in Brazil. On this report, it is described one case of infection after a mammoplasty procedure performed in a private health service in the county of Recife, Pernambuco, Brazil, in 2009. The mycobacteria specie was identified using biochemical tests and sequencing the specific gene rpoB. To treat the infection by Mycobacterium wolinskyi it was necessary to combine antibiotics for a long period of time associated with surgical procedures of the breast abscesses.

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Malik, Richard, Carolyn O'Brien, and Janet Fyfe. "Infections of cats attributable to slow growing or ‘non-culturable’ mycobacteria." Microbiology Australia 30, no. 2 (2009): 92. http://dx.doi.org/10.1071/ma09092.

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Cats are susceptible to a range of different mycobacterial infections. Tuberculosis has not been seen in domestic species living in Australia (including the cat) for decades. Mycobacterial infections most commonly develop in cats subsequent to penetrating injuries (typically inflicted by other cats) that become contaminated with soil or dirt. Most of these infections are caused by rapidly growing mycobacteria, especially Mycobacterium smegmatis and related species, although occasionally other species such as Mycobacterium avium and Mycobacterium ulcerans are involved. In this report we briefly review infections caused by some novel mycobacterial species, which are either impossible or very difficult to grow in vitro using the usual range of liquid and solid media available in reference laboratories. Our understanding of these infections, sometimes referred to as ?feline leprosy-like syndromes?, has increased greatly since the application of molecular techniques and the systematic investigation of affected cats.

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Parish, Tanya, Gretta Roberts, Francoise Laval, Merrill Schaeffer, Mamadou Daffé, and Ken Duncan. "Functional Complementation of the Essential Gene fabG1 of Mycobacterium tuberculosis by Mycobacterium smegmatis fabG but Not Escherichia coli fabG." Journal of Bacteriology 189, no. 10 (March 2, 2007): 3721–28. http://dx.doi.org/10.1128/jb.01740-06.

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ABSTRACT Mycolic acids are a key component of the mycobacterial cell wall, providing structure and forming a major permeability barrier. In Mycobacterium tuberculosis mycolic acids are synthesized by type I and type II fatty acid synthases. One of the enzymes of the type II system is encoded by fabG1. We demonstrate here that this gene can be deleted from the M. tuberculosis chromosome only when another functional copy is provided elsewhere, showing that under normal culture conditions fabG1 is essential. FabG1 activity can be replaced by the corresponding enzyme from the closely related species Mycobacterium smegmatis but not by the enzyme from Escherichia coli. M. tuberculosis carrying FabG from M. smegmatis showed no phenotypic changes, and both the mycolic acids and cell wall permeability were unchanged. Thus, M. tuberculosis and M. smegmatis enzymes are interchangeable and do not control the lengths and types of mycolic acids synthesized.

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Fields, Christopher J., and Robert L. Switzer. "Regulation of pyr Gene Expression in Mycobacterium smegmatis by PyrR-Dependent Translational Repression." Journal of Bacteriology 189, no. 17 (June 29, 2007): 6236–45. http://dx.doi.org/10.1128/jb.00803-07.

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ABSTRACT Regulation of pyrimidine biosynthetic (pyr) genes by a transcription attenuation mechanism that is mediated by the PyrR mRNA-binding regulatory protein has been demonstrated for numerous gram-positive bacteria. Mycobacterial genomes specify pyrR genes and contain obvious PyrR-binding sequences in the initially transcribed regions of their pyr operons, but transcription antiterminator and attenuation terminator sequences are absent from their pyr 5′ leader regions. This work demonstrates that repression of pyr operon expression in Mycobacterium smegmatis by exogenous uracil requires the pyrR gene and the pyr leader RNA sequence for binding of PyrR. Plasmids containing the M. smegmatis pyr promoter-leader region translationally fused to lacZ also displayed pyrR-dependent repression, but transcriptional fusions of the same sequences to a lacZ gene that retained the lacZ ribosome-binding site were not regulated by PyrR plus uracil. We propose that PyrR regulates pyr expression in M. smegmatis, other mycobacteria, and probably in numerous other bacteria by a translational repression mechanism in which nucleotide-regulated binding of PyrR occludes the first ribosome-binding site of the pyr operon.

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Dos Vultos, T., J. Blázquez, J. Rauzier, I. Matic, and B. Gicquel. "Identification of Nudix Hydrolase Family Members with an Antimutator Role in Mycobacterium tuberculosis and Mycobacterium smegmatis." Journal of Bacteriology 188, no. 8 (April 15, 2006): 3159–61. http://dx.doi.org/10.1128/jb.188.8.3159-3161.2006.

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ABSTRACT Mycobacterium tuberculosis and Mycobacterium smegmatis MutT1, MutT2, MutT3, and Rv3908 (MutT4) enzymes were screened for an antimutator role. Results indicate that both MutT1, in M. tuberculosis and M. smegmatis, and MutT4, in M. smegmatis, have that role. Furthermore, an 8-oxo-guanosine triphosphatase function for MutT1 and MutT2 is suggested.

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45

Tyagi, Jaya Sivaswami, and Deepak Sharma. "Mycobacterium smegmatis and tuberculosis." Trends in Microbiology 10, no. 2 (February 2002): 68–69. http://dx.doi.org/10.1016/s0966-842x(01)02296-x.

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NIGOU, Jérôme, and Gurdyal S. BESRA. "Characterization and regulation of inositol monophosphatase activity in Mycobacterium smegmatis." Biochemical Journal 361, no. 2 (January 8, 2002): 385–90. http://dx.doi.org/10.1042/bj3610385.

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Mycobacterium tuberculosis and related members of the genus Mycobacterium contain a number of inositol-based lipids, such as phosphatidylinositol mannosides, lipomannan and lipoarabinomannan. The synthesis of phosphatidylinositol in M. smegmatis is essential for growth and myo-inositol is a key metabolite for mycobacteria. Little is known about the biosynthesis of inositol in mycobacteria and the only known de novo pathway for myo-inositol biosynthesis involves a two-step process. First, cyclization of glucose 6-phosphate to afford myo-inositol 1-phosphate via inositol-1-phosphate synthase and, secondly, dephosphorylation of myo-inositol 1-phosphate by inositol monophosphatase (IMP) to afford myo-inositol. The following report examines IMP activity in M. smegmatis extracts, with regard to pH dependence, bivalent cation re quirement, univalent cation inhibition, regulation by growth and carbon source. We show that IMP activity, which is optimal at the end of the exponential growth phase in Sauton's medium, is Mg2+-dependent. Moreover, IMP activity is inhibited by Li+ and Na+, with Li+ also being able to inhibit growth of M. smegmatis in vivo. This study represents a first step in the delineation of myo-inositol biosynthesis in mycobacteria.

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47

Sharbati, Soroush, Faustine Ravon, Ralf Einspanier, and Jennifer zur Bruegge. "Mycobacterium smegmatis But Not Mycobacterium avium subsp. hominissuis Causes Increased Expression of the Long Non-Coding RNA MEG3 in THP-1-Derived Human Macrophages and Associated Decrease of TGF-β." Microorganisms 7, no. 3 (February 27, 2019): 63. http://dx.doi.org/10.3390/microorganisms7030063.

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Pathogenic mycobacteria are able to persist intracellularly in macrophages, whereas non-pathogenic mycobacteria are effectively combated and eliminated after their phagocytosis. It is known that TGF-β plays an important role in this context. Infection with pathogenic mycobacteria such as Mycobacterium tuberculosis or M. avium leads to production of active TGF-β, which blocks the ability of IFN-γ and TNF-α to inhibit intracellular replication. On the other hand, it is known that the long non-coding RNA (lncRNA) maternally expressed 3 (MEG3) is involved in the regulation of TGF-β. In this study, we show how the infection of THP-1-derived human macrophages with the saprophytic M. smegmatis but not with the facultatively pathogenic M. avium subsp. hominissuis leads to increased MEG3 expression. This is associated with the downregulation of DNA methyltransferases (DNMT) 1 and 3b, which are known to regulate MEG3 expression via promoter hypermethylation. Consequently, we observe a significant downregulation of TGF-β in M. smegmatis-infected macrophages but not in M. avium subsp. hominissuis pointing to lncRNAs as novel mediators of host cell response during mycobacterial infections.

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Meyers, Paul R., William R. Bourn, Lafras M. Steyn, Paul D. van Helden, Albert D. Beyers, and Gordon D. Brown. "Novel Method for Rapid Measurement of Growth of Mycobacteria in Detergent-Free Media." Journal of Clinical Microbiology 36, no. 9 (1998): 2752–54. http://dx.doi.org/10.1128/jcm.36.9.2752-2754.1998.

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We describe a novel, rapid, and inexpensive method for the measurement of growth of Mycobacterium tuberculosis, Mycobacterium bovis, andMycobacterium smegmatis in the presence or absence of detergent. The method, which employs hot NaOH treatment of mycobacterial cells to release total cellular protein, compares favorably with other methods for monitoring mycobacterial growth but is particularly useful for heavily clumped cultures grown in defined minimal medium.

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Madsen, Christian Toft, Lene Jakobsen, and Stephen Douthwaite. "Mycobacterium smegmatis Erm(38) Is a Reluctant Dimethyltransferase." Antimicrobial Agents and Chemotherapy 49, no. 9 (September 2005): 3803–9. http://dx.doi.org/10.1128/aac.49.9.3803-3809.2005.

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ABSTRACT The waxy cell walls of mycobacteria provide intrinsic tolerance to a broad range of antibiotics, and this effect is augmented by specific resistance determinants. The inducible determinant erm(38) in the nontuberculous species Mycobacterium smegmatis confers high resistance to lincosamides and some macrolides, without increasing resistance to streptogramin B antibiotics. This is an uncharacteristic resistance pattern falling between the type I and type II macrolide, lincosamide, and streptogramin B (MLSB) phenotypes that are conferred, respectively, by Erm monomethyltransferases and dimethyltransferases. Erm dimethyltransferases are typically found in pathogenic bacteria and confer resistance to all MLSB drugs by addition of two methyl groups to nucleotide A2058 in 23S rRNA. We show here by mass spectrometry analysis of the mycobacterial rRNA that Erm(38) is indeed an A2058-specific dimethyltransferase. The activity of Erm(38) is lethargic, however, and only a meager proportion of the rRNA molecules become dimethylated in M. smegmatis, while most of the rRNAs are either monomethylated or remain unmethylated. The methylation pattern produced by Erm(38) clarifies the phenotype of M. smegmatis, as it is adequate to confer resistance to lincosamides and 14-member ring macrolides such as erythromycin, but it is insufficient to raise the level of resistance to streptogramin B drugs above the already high intrinsic tolerance displayed by this species.

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Cheadle, Eleanor J., Dearbhaile O'Donnell, Peter J. Selby, and Andrew M. Jackson. "Closely Related Mycobacterial Strains Demonstrate Contrasting Levels of Efficacy as Antitumor Vaccines and Are Processed for Major Histocompatibility Complex Class I Presentation by Multiple Routes in Dendritic Cells." Infection and Immunity 73, no. 2 (February 2005): 784–94. http://dx.doi.org/10.1128/iai.73.2.784-794.2005.

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ABSTRACT Mycobacteria expressing recombinant antigens are already being developed as vaccines against both infections and tumors. Little is known about how dendritic cells might process such antigens. Two different mycobacterial species, the fast-growing Mycobacterium smegmatis and the slow-growing M. bovis M. bovis BCG, were engineered to express a model tumor antigen, the Kb-restricted dominant cytotoxic T-lymphocyte epitope OVA257-264. Recombinant M. bovis BCG but not recombinant M. smegmatis conferred protection to mice challenged with the B16-OVA tumor cell line. We went on to investigate whether the contrast in antitumor efficacy could be due to differences in how dendritic cells process antigen from the two mycobacterial strains for class I presentation. Both strains of mycobacteria caused phenotypic maturation of dendritic cells, but recombinant M. smegmatis infection led to a greater degree of dendritic cell maturation than recombinant M. bovis BCG infection. Antigen from recombinant M. smegmatis was processed and presented as OVA257-264 on Kb molecules by the dendritic cell line DC2.4 but not by bone marrow-derived dendritic cells (BMDC) or splenic dendritic cells. In contrast, antigen from recombinant M. bovis BCG was presented by all three dendritic cell types as long as the mycobacteria were viable. Such presentation was dependent on proteasome function and nascent major histocompatibility complex (MHC) class I molecules in DC2.4 cells but independent of the proteasome and transporter associated with antigen processings (TAP) in BMDC and splenic dendritic cells. These data demonstrate for the first time that antigen vectored by the slow-growing M. bovis BCG but not that vectored by fast-growing, readily destroyed M. smegmatis is processed and presented on MHC class I by in vitro-generated dendritic cells, which has implications for recombinant microbial vaccine development.

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Journal articles on the topic 'Mycobacterium smegmatis'

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