• BMB Reports
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• v.41 no.5
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• pp.363-368
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• 2008

Two genes of temperate mycobacteriophage L5, namely, gp63 and gp64, were hypothesized to be toxic to M. smegmatis. An identical L5 gp64 ortholog (designated hlg1) was cloned from homoimmune mycobacteriophage L1 and characterized at length here. As expected, hlg1 affected the growth of M. smegmatis when overexpressed from a resident plasmid. HLG1 (the protein encoded by hlg1) in fact caused growth retardation of M. smegmatis and the region encompassing its 57-114 C-terminal amino acid residues was found indispensable for its growthretardation activity. Both nucleic acid and protein biosynthesis were severely impaired in M. smegmatis expressing HLG1. Interestingly, HLG1 also affected E. coli almost similarly. This putative delayed early lipoprotein did not participate in the lytic growth of L1.

• BMB Reports
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• v.39 no.2
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• pp.140-144
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• 2006

A deoxyriboendonuclease has been purified to near homogeneity from a fast growing mycobacterium species, M. smegmatis and characterized to some extent. The size of enzyme is about 43 kDa as determined by a denaturing gel analysis. It shows optimum activity at $32^{\circ}C$ in Tris-HCl buffer (pH 7.2) containing 2.5 mM of $MgCl_2$. Both EDTA and $K^+$ but not $Na^+$ inhibit its activity. Evidences show that the enzyme is not a restriction endonuclease but catalyzes the endonucleolytic cleavage of both the double- as well as the single-strand DNA non-specifically. It has been shown that the cleavage by this enzyme generates DNA fragments carrying phosphate groups at 5' ends and hydroxyl group at the 3' ends, respectively. Analysis reveals that no endonuclease having size and property identical to our deoxyriboendonuclease had been purified from M. smegmatis before. The property of our enzymes closely matches with the deoxyriboendonucleases purified from diverse sources including bacteria.

• Journal of Life Science
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• v.24 no.12
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• pp.1330-1338
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• 2014

In Mycobacterium tuberculosis H37Rv 16 adenylyl cyclase (AC) genes have been identified, while 10 AC genes have been found in non-pathogenic Mycobacterium smegmatis. Expression of 6 AC genes (MSMEG_0218, MSMEG_3243, MSMEG_3780, MSMEG_4279, MSMEG_4477, and MSMEG_6154) among 10 AC genes in M. smegmatis was increased when M. smegmatis was subjected to hypoxic growth conditions. On the other hand, only MSMEG_3780 and MSMEG_4279 were slightly induced in the presence of NO. The cAMP levels in cells and culture media were 450- and 9.8-fold increased, respectively, when M. smegmatis was grown under hypoxic conditions relative to those grown aerobically. Intracellular levels of cAMP were increased 5.8-fold on the exposure to NO. The DevSR two-component system is known to be involved in the induction of many genes whose expression is induced under hypoxic conditions and in the presence of NO. Expression of 6 hypoxically induced AC genes was observed to be induced in a devR deletion mutant grown under hypoxic conditions, indicating that the induction of the 6 AC genes under hypoxic conditions is independent of the DevSR two-component system. In order to identify a trans-acting regulatory element that is pertinent in the hypoxic induction of MSMEG_3780, ligand-fishing analysis was performed using the upstream DNA of MSMEG_3780 and MSMEG_5136 protein was identified.

• Journal of Microbiology and Biotechnology
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• v.29 no.8
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• pp.1221-1229
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• 2019

Mycobacterium tuberculosis, a causative pathogen of tuberculosis (TB), still threatens human health worldwide. To find a novel drug to eradicate this pathogen, we tested taurine-5-bromosalicylaldehyde Schiff base (TBSSB) as an innovative anti-mycobacterial drug using Mycobacterium smegmatis as a surrogate model for M. tuberculosis. We investigated the antimicrobial activity of TBSSB against M. smegmatis by plotting growth curves, examined the effect of TBSSB on biofilm formation, observed morphological changes by scanning electron microscopy and transmission electron microscopy, and detected differentially expressed proteins using two-dimensional gel electrophoresis coupled with mass spectrometry. TBSSB inhibited mycobacterial growth and biofilm formation, altered cell ultrastructure and intracellular content, and inhibited cell division. Furthermore, M. smegmatis adapted itself to TBSSB inhibition by regulating the metabolic pathways and enzymatic activities of the identified proteins. NDMA-dependent methanol dehydrogenase, NAD(P)H nitroreductase, and amidohydrolase AmiB1 appear to be pivotal factors to regulate the M. smegmatis survival under TBSSB. Our dataset reinforced the idea that Schiff base-taurine compounds have the potential to be developed as novel anti-mycobacterial drugs.

• Korean Journal of Microbiology
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• v.24 no.2
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• pp.147-153
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• 1986

Viable potential of Mycobacterium smegmatis, a slow grower in vitro cultivation and of M. leprae, an obligate intracellular parasitic bacterium, which can not be cultured yet in vitro was assessed by fluorospectrophotometry. Bacterial cells in different numbers and under various physiological status were incubater with fluorescein diacetate(FDA). After an incubation of the bacterial preparations with FDA at specified conditions, amount of fluorescein inside bacteria was measured by a fluorospectrophotometer at 470nm and 510nm of excitation and emission wavelengths, respectively. Fluorounit given by such bacteria showed a correlation with assessment of viability of the same preparations made by other methods, such as optical density and colony forming units of M. smegmatis and intracellular ATP content of M. leprae. The possible use of fluorospectrophotometry in assessing viability or physiological potential of bacteria, particularly intracellular parasites and fastidious organisms to culture in vitro is discussed in relation to other methods.

• BMB Reports
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• v.32 no.5
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• pp.461-467
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• 1999

Recombinant Mycobacterium smegmatis expressing ovalbumin was used to immunize C57BL/6(H-$2^b$) mice, and the humoral immunity against recombinant ovalbumin was analyzed. Antibodies were purified by denatured ovalbumin-conjugated affinity chromatography. The epitopes of the antibodies were screened with a random peptide library displayed on the tip of fUSE5 filamentous phage pIII minor coat proteins. Two peptides, IRLADR and SPGAEV, were selected predominantly by the recognition of purified antibodies using biopanning methods. The composition of the peptide sequence with the primary structure of OVA revealed that the peptide sequence analogizes to INEAGR, part of the $^{323}ISQAVHAAHAEINEAGR^{339}$ sequence previously reported as the antigenic determinant for murine Band also Th cell epitopes (I-$A^d$ binding). Also, the structures of these mimotopes obtained from restrained molecular dynamic computations resulted in the formation of a $\beta$-turn proven to be a secondary structure of the parent peptide within the ovalbumin molecule, enabling us to confirm the structural similarity. This study demonstrates that immunization with recombinant M. smegmatis can generate neutralizing antibodies identical with those induced by the administration of natural antigenic proteins and supports the potential use of mycobacteria as vaccine delivery vehicles.

• Journal of Microbiology and Biotechnology
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• v.27 no.8
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• pp.1483-1490
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• 2017

In this study, silver nanoparticles (AgNPs) were synthesized by the citrate reduction process and, with the assistance of n-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, were successfully loaded with the macromolecular drug vancomycin (VAM) to form AgNP-VAM bioconjugates. The synthesized AgNPs, VAM, and AgNP-VAM conjugate were characterized by UV-visible spectroscopy, zeta potential analysis, confocal microscopy, and transmission electron microscopy. The effect of loading VAM onto AgNPs was investigated by testing the internalization of the bioconjugate into Mycobacterium smegmatis. After treatment with the AgNP-VAM conjugate, the bacterial cells showed a significant decrease in UV absorption, indicating that loading of the VAM on AgNPs had vastly improved the drug's internalization compared with that of AgNPs. All the experimental assessments showed that, compared with free AgNPs and VAM, enhanced internalization had been successfully achieved with the AgNP-VAM conjugate, thus leading to significantly better delivery of the macromolecular drug into the M. smegmatis cell. The current research provides a new potential drug delivery system for the treatment of mycobacterial infections.

• Journal of Life Science
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• v.20 no.6
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• pp.853-860
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• 2010

The DevSR two-component system is a major regulatory system involved in redox sensing in Mycobacterium smegmatis. The DevSR system consists of the DevS histidine kinase and its cognate DevR response regulator. When exposed to hypoxic conditions, the DevS histidine kinase is activated to phosphorylate the DevR response regulator, leading to the transcriptional activation of the DevR regulation. The ligand-binding state of the heme embedded in the N-terminal GAF domain of DevS determines the kinase activity of DevS. In this study, we demonstrated that the redox-responsive cysteine (C547) in the C-terminal kinase domain is involved in the redox-dependent control of DevS kinase activity. The formation of an intersubunit disulfide bond between the C547 residues in the presence of $O_2$ led to inactivation of DevS kinase activity. The reduction of the oxidized DevS with reductants such as $\beta$-mercaptoethanol and dithiothreitol resulted in the restoration of DevS kinase activity. It was demonstrated in vivo by complementation test that the substitution of C547 to alanine partially impaired the sensory function of DevS in M. smegmatis.

• BMB Reports
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• v.37 no.4
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• pp.487-492
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• 2004

The extent of codon usage in the protein coding genes of the mycobacteriophage, Bxz1, and its plating bacteria, M. smegmatis, were determined, and it was observed that the codons ending with either G and / or C were predominant in both the organisms. Multivariate statistical analysis showed that in both organisms, the genes were separated along the first major explanatory axis according to their expression levels and their genomic GC content at the synonymous third positions of the codons. The second major explanatory axis differentiates the genes according to their genome type. A comparison of the relative synonymous codon usage between 20 highly- and 20 lowly expressed genes from Bxz1 identified 21 codons, which are statistically over represented in the former group of genes. Further analysis found that the Bxz1- specific tRNA species could recognize 13 out of the 21 over represented synonymous codons, which incorporated 13 amino acid residues preferentially into the highly expressed proteins of Bxz1. In contrast, seven amino acid residues were preferentially incorporated into the lowly expressed proteins by 10 other tRNA species of Bxz1. This analysis predicts for the first time that the Bxz1-specific tRNA species modulates the optimal expression of its proteins during development.

• Journal of Microbiology and Biotechnology
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• v.29 no.9
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• pp.1401-1411
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• 2019

Mycobacterial cell walls comprise thick and diverse lipids and glycolipids that act as a permeability barrier to antibiotics or other chemical agents. The use of OH radicals from a non-thermal plasma jet (NTPJ) for the inactivation of mycobacteria in aqueous solution was adopted as a novel approach. Addition of water vapor in a nitrogen plasma jet generated OH radicals, which converted to hydrogen peroxide ($H_2O_2$) that inactivated non-pathogenic Mycobacterium smegmatis and pathogenic Mycobacterium tuberculosis H37Rv. A stable plasma plume was obtained from a nitrogen plasma jet with 1.91 W of power, killing Escherichia coli and mycobacteria effectively, whereas addition of catalase decreased the effects of the former. Mycobacteria were more resistant than E. coli to NTPJ treatment. Plasma treatment enhanced intracellular ROS production and upregulation of genes related to ROS stress responses (thiolrelated oxidoreductases, such as SseA and DoxX, and ferric uptake regulator furA). Morphological changes of M. smegmatis and M. tuberculosis H37Rv were observed after 5 min treatment with $N_2+H_2O$ plasma, but not of pre-incubated sample with catalase. This finding indicates that the bactericidal efficacy of NTPJ is related to the toxicity of OH and $H_2O_2$ radicals in cells. Therefore, our study suggests that NTPJ treatment may effectively control pulmonary infections caused by M. tuberculosis and nontuberculous mycobacteria (NTM) such as M. avium or M. abscessus in water.