• BMB Reports
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• 제41권5호
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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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• 제39권2호
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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.

• 생명과학회지
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• 제24권12호
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• pp.1330-1338
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• 2014

결핵균인 Mycobacterium tuberculosis H37Rv에서는 16개의 adenylyl cyclase (AC) 유전자가, 비병원성인 M. smegmatis에서는 10개의 AC 유전자가 발견되었다. M. smegmatis에서 발견된 10개의 AC 유전자들 중 MSMEG_0228, MSMEG_3243, MSMEG_3780, MSMEG_4279, MSMEG_4477, MSMEG_6154 유전자들의 발현은 저산소 조건에서 유도되었다. 반면에 M. smegmatis가 nitric oxide (NO)에 노출이 되면 MSMEG_3780과 MSMEG_4279 유전자의 발현이 유도되었다. 유산소 조건에서 키운 M. smegmatis와 비교할 때, 저산소 조건에서 성장한 M. smegmatis 세포 내부와 성장 배지 내의 cAMP 양은 각각 450배와 9.8배 증가하였다. M. smegamtis가 NO에 노출이 되면 세포 내의 cAMP 농도는 5.8배 증가하였다. DevSR two-component system은 M. smegmatis가 저산소 조건이나 NO에 노출되었을 때 많은 유전자의 발현을 유도하는 주요 조절계로 알려져 있는데, 저산소 조건에서 발현이 유도된 6개의 AC 유전자의 발현이 M. smegmatis devR mutant에서도 wild type에서와 마찬가지로 저산소 조건에서 유도됨이 관찰되었다. 이 결과는 저산소 조건에서 발현이 유도되는 6개의 AC 유전자들이 DevSR two-component system에 의해 조절되지 않음을 나타내고 있다. 저산소 조건에서 발현이 가장 많이 유도된 MSMEG_3780 유전자의 발현조절에 관련이 있는 조절자를 발굴하기 위해 ligand fishing 분석을 수행하였고, 후보 조절자로서 MSMEG_5136을 발굴하였다.

• Journal of Microbiology and Biotechnology
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• 제29권8호
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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.

• 미생물학회지
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• 제24권2호
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• pp.147-153
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• 1986

나균(M. leprae)을 비롯한 항산균(mycobacterium)의 시험관내 생리력 또는 생명력평가법(in vitro assessment of physiological potential or viability)으로 형광분광측정법(fluorospectrophotometry)를 고안하였다. 균액을 비형광성의 fluorescein diacetate(FDA)로 처리, 균체의 생대사능에 의해 FDA로부터 유리된 체내 fluorescein 량을 Aminco-Bowman 형광분광기로 측정함으로 균체의 생리능을 fluorounit로 표기해 보았다. Fluorounit로 표기된 균체의 생명력을 균배양의 광학밀도(optical density, colony forming unit, 체내 ATP 량 intracellular ATP content)등으로 표기되는 항산균의 생명력고 비교 검토함으로 형광분광법에 의한 시험관내 항산균의 생명력 평가법의 적합성을 살펴보았다. 형광분광측정에 의한 생명도의 평가는 객관성을 띤 기기정량법으로 조작이 간단하고 신속하게 결과를 얻을 수 있어 시험관내 배양의 속도가 완만한 균이나(slow growing mycobacteria, I.e.M. lerpae, Listeriae sp)등의 생명력의 상대적 평가에 활용될 수 있다고 사료된다.

• BMB Reports
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• 제32권5호
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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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• 제27권8호
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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.

• 생명과학회지
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• 제20권6호
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• pp.853-860
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• 2010

DevSR two-component system은 Mycobacterium smegmatis의 redox sensing에 관련된 주요한 regulatory system이다. DevSR system은 DevS histidine kinase와 DevR response regulator로 구성되어 있다. 저산소 조건에서 DevS histidine kinase는 활성화되어 DevR response regulator를 인산화 시키고, 인산화된 DevR response regulator는 DevR regulon의 transcriptional activator로 작용한다. DevS의 kinase activity는 DevS의 N-terminal에 위치한 GAF domain에 존재하는 heme의 ligand-binding state에 의해 결정된다. 본 연구에서는 C-terminal kinase domain의 redox-responsive cysteine (C547)이 DevS kinase activity의 redox-dependent control과 연관이 있음을 밝혔다. 산소가 존재할 때, C547 residue 사이의 disulfide bond의 형성은 DevS kinase activity를 불활성화 시킨다. $\beta$-mercaptoethanol과 dithiothreitol과 같은 환원제를 이용하여 산화된 DevS를 환원시켰을 때, DevS kinase activity가 복원된 것이 관찰되었다. 또한, C547을 alanine으로 치환했을 때, M. smegmatis의 DevS의 sensory 기능을 부분적으로 손상되는 것이 complementation 실험을 통해 in vivo 상에서 증명되었다.

• BMB Reports
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• 제37권4호
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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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• 제29권9호
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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.