AbmR (Rv1265) is a novel transcription factor of Mycobacterium tuberculosis that regulates host cell association and expression of the non‐coding small RNA Mcr11

Abstract

SummaryGene regulatory networks used by Mycobacterium tuberculosis (Mtb) during infection include many genes of unknown function, confounding efforts to determine their roles in Mtb biology. Rv1265 encodes a conserved hypothetical protein that is expressed during infection and in response to elevated levels of cyclic AMP. Here, we report that Rv1265 is a novel auto‐inhibitory ATP‐binding transcription factor that upregulates expression of the small non‐coding RNA Mcr11, and propose that Rv1265 be named ATP‐binding mcr11 regulator (AbmR). AbmR directly and specifically bound DNA, as determined by electrophoretic mobility shift assays, and this DNA‐binding activity was enhanced by AbmR’s interaction with ATP. Genetic knockout of abmR in Mtb increased abmR promoter activity and eliminated growth phase‐dependent increases in mcr11 expression during hypoxia. Mutagenesis identified arginine residues in the carboxy terminus that are critical for AbmR’s DNA‐binding activity and gene regulatory function. Limited similarity to other DNA‐ or ATP‐binding domains suggests that AbmR belongs to a novel class of DNA‐ and ATP‐binding proteins. AbmR was also found to form large organized structures in solution and facilitate the serum‐dependent association of Mtb with human lung epithelial cells. These results indicate a potentially complex role for AbmR in Mtb biology.

Small RNA Mcr11 requires the transcription factor AbmR for stable expression and regulates genes involved in the central metabolism of Mycobacterium tuberculosis

“…Thus, we added a copy of the Mtb abmR locus upstream of the P mcr11 : GFPv fusion sequences and re‐tested the activity of P mcr11 in response to growth phase in Msm. The addition of abmR significantly increased P mcr11 activity in Msm, and rendered P mcr11 activity responsive to growth phase (Figure e), as previously observed in BCG and Mtb (Girardin et al, ). Inclusion of abmR also significantly increased the amount of stable Mcr11 detected by Northern blot in Msm (Figure d), indicating that Mcr11 is unstable even when abundantly transcribed in Msm.…”

“…We also demonstrated that transcriptional termination and stable production of Mcr11 are enhanced by extended native sequence 3′ to mcr11 along with the product of the divergently transcribed adjacent gene, abmR (Figure ). The regulation of central metabolism in Mtb is consistent with mcr11 expression profiles in response to growth phase and in vivo infection (Girardin et al, ; Pelly et al, ). However, the additional requirements for TB complex specific factors for stable Mcr11 expression were unexpected, and may have broader implications for understanding sRNA expression in Mtb.…”

“…Mcr11 expression varies across growth phases in TB complex mycobacteria (DiChiara et al, ; Girardin et al, ), so we examined the effect of growth phase on mcr11 expression in Msm. Fluorescence from the GFPv transcriptional reporters was measured at mid‐log and late stationary phases.…”

“…Previously, we showed that the divergently expressed DNA‐binding protein AbmR is a growth phase‐responsive activator of mcr11 expression (Girardin et al, ). Although the Msm orthologue ( MSMEG_5010) of abmR displays both high amino acid sequence identity (68.75%) and similarity (83.51%) to AbmR (Girardin et al, ), we reasoned that abmR could have functionality that the Msm orthologue lacks. Thus, we added a copy of the Mtb abmR locus upstream of the P mcr11 : GFPv fusion sequences and re‐tested the activity of P mcr11 in response to growth phase in Msm.…”

“…Constructs including the mcr11 promoter, Mcr11 sequence and various potential intrinsic terminator sequences were fused to the reporter green fluorescent protein Venus ( GFPv ) on an integrating plasmid as previously described (Girardin et al, ). A GFPv fluorescence assay was used to measure promoter activity and read‐through of the putative intrinsic terminators of Mcr11 in Msm, BCG and Mtb.…”