Journal: Frontiers in Microbiology

Article Title: Localization of Mycobacterium tuberculosis topoisomerase I C-terminal sequence motif required for inhibition by endogenous toxin MazF4

doi: 10.3389/fmicb.2022.1032320

Figure Lengend Snippet: Assay for growth complementation of Escherichia coli AS17 with temperature sensitive topA. Ten-fold serial dilution of E. coli AS17 transformed with indicated plasmids expressing recombinant topoisomerase I (pLIC-ETOP and pLIC-MTOP), MazF4 toxin (pBAD-Rv1495) or control vectors (pLIC-HK and pBAD/Thio) were spotted on LB agar plates with antibiotics and 0.2% arabinose. The set of duplicated plates was incubated at 30°C (left) and 42°C (right).

Article Snippet: The clones were isolated as transformants of Escherichia coli NEB ® 5-alpha competent cells (from New England BioLabs).

Techniques: Serial Dilution, Transformation Assay, Expressing, Recombinant, Incubation

Journal: Frontiers in Microbiology

Article Title: Localization of Mycobacterium tuberculosis topoisomerase I C-terminal sequence motif required for inhibition by endogenous toxin MazF4

doi: 10.3389/fmicb.2022.1032320

Figure Lengend Snippet: Complementation of AS17 by C-terminal truncation mutants of Mtb TopA. (A) Schematic diagram of subdomains found in full length Mtb TopA and mutants truncated at residue 910 and 840. Complementation of E. coli AS17 by pLIC-MTOP-840 t (B) and pLIC-MTOP-910 t (C) truncated mutants in the presence of pBAD/Thio or pBAD-Rv1495 was compared with complementation by pLIC-MTOP with full length Mtb TopA.

Article Snippet: The clones were isolated as transformants of Escherichia coli NEB ® 5-alpha competent cells (from New England BioLabs).

Techniques:

Journal: Frontiers in Microbiology

Article Title: Localization of Mycobacterium tuberculosis topoisomerase I C-terminal sequence motif required for inhibition by endogenous toxin MazF4

doi: 10.3389/fmicb.2022.1032320

Figure Lengend Snippet: Model for the relaxation of supercoiled DNA by bacterial topoisomerase I based on the crystal structures of E. coli and Mycobacteria topoisomerase I. (i) Apo enzyme; (ii) C-terminal domains (green) recognizes ssDNA region in unwound DNA duplex as T-strand (red); (iii) ssDNA or G-strand (yellow) binds the N-terminal domains (blue); (iv) Active site tyrosine (red circle) becomes accessible; (v) Cleavage of the G-strand and gate opening with T-strand DNA approaching toroid hole; (vi) Passage of T-strand inside the toroid; (vii) Gate closing and trapping of T-strand; (viii) Religation of the G-strand; (ix) Gate opening and release of dsDNA. Figure reproduced from .

Article Snippet: The clones were isolated as transformants of Escherichia coli NEB ® 5-alpha competent cells (from New England BioLabs).

Techniques: