Biosensor-integrated transposon mutagenesis reveals rv0158 as a coordinator of redox homeostasis in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) is evolutionarily equipped to resist exogenous reactive oxygen species (ROS) but shows vulnerability to an increase in endogenous ROS (eROS). Since eROS is an unavoidable consequence of aerobic metabolism, understanding how Mtb manages eROS levels is essential yet needs to be characterized. By combining the Mrx1-roGFP2 redox biosensor with transposon mutagenesis, we identified 368 genes (redoxosome) responsible for maintaining homeostatic levels of eROS in Mtb. Integrating redoxosome with a global network of transcriptional regulators revealed a hypothetical protein (Rv0158) as a critical node managing eROS in Mtb. Disruption of rv0158 (rv0158 KO) impaired growth, redox balance, respiration, and metabolism of Mtb on glucose but not on fatty acids. Importantly, rv0158 KO exhibited enhanced growth on propionate, and the Rv0158 protein directly binds to methylmalonyl-CoA, a key intermediate in propionate catabolism. Metabolite profiling, ChIP-Seq, and gene-expression analyses indicate that Rv0158 manages metabolic neutralization of propionate toxicity by regulating the methylcitrate cycle. Disruption of rv0158 enhanced the sensitivity of Mtb to oxidative stress, nitric oxide, and anti-TB drugs. Lastly, rv0158 KO showed poor survival in macrophages and persistence defect in mice. Our results suggest that Rv0158 is a metabolic integrator for carbon metabolism and redox balance in Mtb.

A cytosine-patch sequence motif identified in the conserved region of lincRNA-p21 interacts with the KH3 domain of hnRNPK.

Long Intergenic Non-coding RNAs (lincRNAs) are the largest class of long non-coding RNAs in eukaryotes, originating from the genome's intergenic regions. A ∼4 â€‹kb long lincRNA-p21 is derived from a transcription unit next to the p21/Cdkn1a gene locus. LincRNA-p21 plays regulatory roles in p53-dependent transcriptional and translational repression through its physical association with proteins such as hnRNPK and HuR. It is also involved in the aberrant gene expression in different cancers. In this study, we have carried out a bioinformatics-based gene analysis and annotation of lincRNA-p21 to show that it is highly conserved in primates and identified two conserved domains in its sequence at the 5' and 3' terminal regions. hnRNPK has previously been shown to interact specifically with the 5' conserved region of lincRNA-p21. hnRNPK is known to bind preferentially to the pyrimidine-rich (poly C) nucleotide sequences in RNAs. Interestingly, we observed a single occurrence of a cytosine-rich patch (C-patch) consisting of a CUCCCGC sequence in the 5' conserved region of human lincRNA-p21, making it a putative hnRNPK binding motif. Using NMR and ITC experiments, we showed that the single-stranded C-patch containing RNA sequence motif interacts specifically with the KH3 domain of hnRNPK.