Mce2R/Rv0586 of Mycobacterium tuberculosis is the functional homologue of FadR E. coli

Abstract

Lipid metabolism is critical to Mycobacterium tuberculosis survival and infection. Unlike Escherichia coli, which has a single FadR, the M. tuberculosis genome encodes five proteins of the FadR sub-family. While the role of E. coli FadR as a regulator of fatty acid metabolism is well known, the definitive functions of M. tuberculosis FadR proteins are still under investigation. An interesting question about the M. tuberculosis FadRs remains open: which one of these proteins is the functional homologue of E. coli FadR? To address this, we have applied two different approaches. The first one was the bioinformatics approach and the second one was the classical molecular genetic approach involving complementation studies. Surprisingly, the results of these two approaches did not agree. Among the five M. tuberculosis FadRs, Rv0494 shared the highest sequence similarity with FadR and Rv0586 was the second best match. However, only Rv0586, but not Rv0494, could complement E. coli ∆fadR, indicating that Rv0586 is the M. tuberculosis functional homologue of FadR. Further studies showed that both regulators, Rv0494 and Rv0586, show similar responsiveness to LCFA, and have conserved critical residues for DNA binding. However, analysis of the operator site indicated that the inter-palindromic distance required for DNA binding differs for the two regulators. The differences in the binding site selection helped in the success of Rv0586 binding to fadB upstream over Rv0494 and may have played a critical role in complementing E. coli ∆fadR. Further, for the first time, we report the lipid-responsive nature of Rv0586.

The Escherichia coli FadR transcription factor: Too much of a good thing?

“…However, the parochial γ‐proteobacterial view of FadR distribution has been overturned by the Mce2R/Rv0586 protein of Mycobacterium tuberculosis . Expression of this protein in E. coli blocks growth of a ∆fadR strain on decanoate (Yousuf et al, 2018). This result argues that the M. tuberculosis protein recognizes the E. coli FadR operator sequence and that its native operator must have a similar sequence.…”

“…This result argues that the M. tuberculosis protein recognizes the E. coli FadR operator sequence and that its native operator must have a similar sequence. This is the case, the Mce2R/Rv0586 protein binds to an extended version of the E. coli FadR binding site and several DNA binding residues of E. coli FadR are both conserved in the mycobacterial protein and were shown to be required for DNA binding (Yousuf et al, 2018). Finally, long‐chain acyl‐CoAs at physiologically reasonable concentrations (25‒50 μM) prevented DNA binding by Mce2R/Rv0586 demonstrating it to be a valid functional homolog of E. coli FadR (Yousuf et al, 2018).…”

“…This is the case, the Mce2R/Rv0586 protein binds to an extended version of the E. coli FadR binding site and several DNA binding residues of E. coli FadR are both conserved in the mycobacterial protein and were shown to be required for DNA binding (Yousuf et al, 2018). Finally, long‐chain acyl‐CoAs at physiologically reasonable concentrations (25‒50 μM) prevented DNA binding by Mce2R/Rv0586 demonstrating it to be a valid functional homolog of E. coli FadR (Yousuf et al, 2018). M. tuberculosis encodes a second FadR homolog, Rv0494 which has a greater sequence similarity to E. coli FadR than does Rv0586 (24% vs 20%) and is also a DNA‐binding protein whose binding is disrupted by long‐chain acyl‐CoAs.…”

“…M. tuberculosis encodes a second FadR homolog, Rv0494 which has a greater sequence similarity to E. coli FadR than does Rv0586 (24% vs 20%) and is also a DNA‐binding protein whose binding is disrupted by long‐chain acyl‐CoAs. However, Rv0494 recognizes an operator unrelated to the E. coli operators and thus was unable to complement the E. coli ∆fadR strain (Yousuf et al, 2018).…”

Ectopic Expression of Rv0023 Mediates Isoniazid/Ethionamide Tolerance via Altering NADH/NAD+ Levels in Mycobacterium smegmatis

“…Cells were grown in LB broth at 37 • C till mid-log phase, and then 1 mM IPTG (isopropyl 1-thio-β-D-galactopyranoside) was added to induce the expression of protein. Cells were grown for another 4 h at 37 • C, after which cells were harvested and purified using Ni-NTA affinity chromatography as described earlier (Yousuf et al, 2018). The purity of recombinant protein was analyzed…”