DNA Isolation from Chloroform/Methanol-Treated Mycobacterial Cells without Lysozyme and Proteinase K

Mve-Obiang, Armand; Mestdagh, Marian; Portaels, Françoise

doi:10.2144/01302bm07

Cited by 7 publications

(6 citation statements)

References 5 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The resistance to TBA161-C was determined by testing the susceptibility of strains to TBA161-C using a REMA assay. Genomic DNA extraction of TBA161-C-resistant and parental mycobacterial strains was performed using phenol/chloroform/isoamyl-alcohol extraction as described previously ( Mve-Obiang et al, 2001 ). Whole-genome sequencing of genomic DNA from parental wild-type M. marinum - tdTomato , three TBA161-C-resistant M. marinum - tdTomato strains and TBA161-C-resistant Mtb strain were outsourced to Beijing Novogene Bioinformatics Technology (Novogene, China) using Illumina sequencing technology.…”

Section: Methodsmentioning

confidence: 99%

“…The resistance to TBA161-C was determined by testing the susceptibility of strains to TBA161-C using the REMA assay. Genomic DNA extraction of TBA161-C resistant and parental mycobacterial strains was done using phenol/chloroform/isoamyl-alcohol extraction as described previously (Mve-Obiang et al, 2001).…”

Section: Generation and Characterization Of Spontaneous Tba161-c Resistant Mutantsmentioning

confidence: 99%

See 1 more Smart Citation

An anti-tuberculosis compound screen using a zebrafish infection model identifies an aspartyl-tRNA synthetase inhibitor

Habjan

Gallant

et al. 2021

Disease Models &Amp; Mechanisms

View full text Add to dashboard Cite

Finding new anti-tuberculosis compounds with convincing in vivo activity is an ongoing global challenge to fight the emergence of multi-drug resistant Mycobacterium tuberculosis isolates. In this work, we exploited the medium-throughput capabilities of the zebrafish embryo infection model with Mycobacterium marinum as a surrogate for M. tuberculosis. Using a representative set of clinically established drugs, we demonstrate that this model could be predictive and selective for antibiotics that can be administered orally. We further used the zebrafish-infection model to screen 240 compounds from an anti-TB hit library for their in vivo activity and identified 14 highly active compounds. One of the most active compounds was the tetracyclic compound TBA161, which was studied in more detail. Analysis of resistant mutants revealed point mutations in aspS (rv2572c), encoding an aspartyl-tRNA synthetase. The target was genetically confirmed, and molecular docking studies propose possible binding of TBA161 in a pocket adjacent to the catalytic site. This study showed that the zebrafish-infection model is suitable to rapidly identify promising scaffolds with in vivo activity.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Generation and Characterization Of Spontaneous Tba161-c Resistant Mutantsmentioning

confidence: 99%

An anti-tuberculosis compound screen using a zebrafish infection model identifies an aspartyl-tRNA synthetase inhibitor

Habjan

Gallant

et al. 2021

Disease Models &Amp; Mechanisms

View full text Add to dashboard Cite

show abstract

“…Genomic DNA was extracted from eight of the CEI (01-627, 01-628, 01-629, 01-632, 01-633, 01-636, 01-664, and 01-665) by a DNA extraction method for mycobacteria previously described (12). PCR products from the 16S rRNA primers were transformed into Escherichia coli with the TA TOPO cloning kit (Invitrogen, Carlsbad, Calif.), and transformants were selected by 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-Gal)-mediated bluewhite screening and ampicillin resistance.…”

Section: Methodsmentioning

confidence: 99%

Analysis of Mycobacterium Species for the Presence of a Macrolide Toxin, Mycolactone

et al. 2004

View full text Add to dashboard Cite

Mycobacterium ulcerans is an environmental organism which is responsible for the disease Buruli ulcer, a necrotizing skin disease emerging in west Africa. M. ulcerans produces the polyketide-derived macrolide mycolactone, which is required for the immunosuppression and tissue damage which characterizes Buruli ulcer. We have extracted lipids from the cell envelope and culture filtrate from 52 isolates of Mycobacterium species, analyzed them with thin-layer chromatography, and tested them in a murine fibroblast cell line (L929) cytotoxicity assay to investigate whether these mycobacterial species produce mycolactone. For these studies chloroform-methanol (2:1, vol/vol) extracts were prepared from representative fast-and slow-growing mycobacterial species. Isolates tested included 16 uncharacterized, slow-growing, environmental mycobacterial species isolated from areas in which M. ulcerans infection is endemic. Although several strains of mycobacteria studied produced cytopathic lipids, none of these produced a phenotype on cultured cells consistent with that produced by mycolactone. Two mycobacterial species, M. scrofulaceum and M. kansasii, and eight of the environmental mycobacterial isolates contained cell-associated lipids cytopathic to fibroblasts at concentrations of 33 to 1,000 g/ml. In contrast, mycolactone produces cytotoxicity at less than 2 ng/ml. Analysis of 16S rRNA sequences from the eight environmental isolates suggests that these are novel mycobacterial species. Results from these studies suggest that, although production of cytopathic lipids is relatively common among mycobacterial species, the production of mycolactone as a cell-associated or secreted molecule appears so far to be restricted to M. ulcerans.

show abstract

“…DNA was isolated from whole cells after a chloroform/methanol wash with a disruption solution of guanidine thiocyamate, sarkosyl and mercaptoethanol as described in Mve-Obiang et al [16]. The purity of DNA was assessed by The Broad Institute using the Quant-iT™ dsDNA Assay High Sensitivity Kit (Invitrogen, Carlsbad, CA) and according to the manufacturer's protocol.…”

Section: Genome Sequencing and Annotationmentioning

confidence: 99%

High quality draft genome sequence of Segniliparus rugosus CDC 945T= (ATCC BAA-974T)

Earl

Desjardins

FitzGerald

et al. 2011

Stand. Genomic Sci.

View full text Add to dashboard Cite

Segniliparus rugosus represents one of two species in the genus Segniliparus, the sole genus in the family Segniliparaceae. A unique and interesting feature of this family is the presence of extremely long carbon-chain length mycolic acids bound in the cell wall. S. rugosus is also a medically important species because it is an opportunistic pathogen associated with mammalian lung disease. This report represents the second species in the genus to have its genome sequenced. The 3,567,567 bp long genome with 3,516 protein-coding and 49 RNA genes is part of the NIH Roadmap for Medical Research, Human Microbiome Project.

show abstract

DNA Isolation from Chloroform/Methanol-Treated Mycobacterial Cells without Lysozyme and Proteinase K

Cited by 7 publications

References 5 publications

An anti-tuberculosis compound screen using a zebrafish infection model identifies an aspartyl-tRNA synthetase inhibitor

An anti-tuberculosis compound screen using a zebrafish infection model identifies an aspartyl-tRNA synthetase inhibitor

Analysis of Mycobacterium Species for the Presence of a Macrolide Toxin, Mycolactone

High quality draft genome sequence of Segniliparus rugosus CDC 945T= (ATCC BAA-974T)

Contact Info

Product

Resources

About