ABSTRACT: We have performred RNA-seq analysis of WT, ∆phoP and ∆sigH Mtb H37Rv under normal and redox (Diamide) stress, to investigate the role of PhoP and SigH in maintaning the redox homoeostasis of bacterium. RNA was extracted from exponentially growing mycobacterial cells in Middlebrook 7H9 media. Briefly, 25 ml of bacterial culture was grown to mid-log phase (OD600= 0.4 to 0.6) and combined with 40 ml of 5 M guanidinium thiocyanate solution containing 1% β-mercaptoethanol and 0.5% Tween 80. Cells were pelleted by centrifugation, and lysed by re-suspending in 1 ml Trizol (Ambion) in the presence of Lysing Matrix B (100 µm silica beads; MP Bio) using a FastPrep-24 bead beater (MP Bio) at a speed setting of 6.0 for 30 seconds. The procedure was repeated for 2-3 cycles with incubation on ice in between pulses. Next, cell lysates were centrifuged at 13000 rpm for 10 minutes; supernatant was collected and processed for RNA isolation using Direct-ZolTM RNA isolation kit (ZYMO) as per manufacturer’s recommendation. Following extraction, RNA was treated with DNAse I (Promega) to degrade contaminating DNA, and integrity was assessed using a Nanodrop (ND-1000, Spectrophotometer). RNA samples were further checked for intactness of 23S and 16S rRNA using formaldehyde-agarose gel electrophoresis, and Qubit fluoremeter (Invitrogen). RNA integrity was checked using Agilent 2200 Tape Station system (Agilent Technologies). Library construction, RNA-sequencing and data analysis have been carried out by Agrigenome Labs Private Limited (Cochin), India. The main purpose of this study is to understand how mycobacteria can sense numerous stress conditions and mount an appropriate stress response. Although recent evidence suggests that at low pH M. tuberculosis encounters reductive stress, the mechanism of integrated regulation of stress response remains unknown. Unexpectedly, we find that PhoP contributes to mycothiol level and a PhoP-depleted bacilli shows enhanced susceptibility to redox stress. Because SigH is known to control expression of redox inducible genes, we probed whether previously-reported PhoP-SigH interaction accounts for mycobacterial redox stress response. Our results suggest that while PhoP controls pH homeostasis via its interaction with SigE, SigH-dependent PhoP expression, but not PhoP -SigH interaction, and direct recruitment of SigH, but not PhoP controls expression of mycobacterial thioredoxin genes. Together, these results uncover novel stress-specific interaction events of sigma factors and PhoP or lack thereof, as the underlying mechanisms of an adaptive programme, which couples low pH conditions and mycobacterial thiol homeostasis. The main purpose of this study is to understand how mycobacteria can sense numerous stress conditions and mount an appropriate stress response. Although recent evidence suggests that at low pH M. tuberculosis encounters reductive stress, the mechanism of integrated regulation of stress response remains unknown. Unexpectedly, we find that PhoP contributes to mycothiol level and a PhoP-depleted bacilli shows enhanced susceptibility to redox stress. Because SigH is known to control expression of redox inducible genes, we probed whether previously-reported PhoP-SigH interaction accounts for mycobacterial redox stress response. Our results suggest that while PhoP controls pH homeostasis via its interaction with SigE, SigH-dependent PhoP expression, but not PhoP -SigH interaction, and direct recruitment of SigH, but not PhoP controls expression of mycobacterial thioredoxin genes. Together, these results uncover novel stress-specific interaction events of sigma factors and PhoP or lack thereof, as the underlying mechanisms of an adaptive programme, which couples low pH conditions and mycobacterial thiol homeostasis.
