The RosR transcription factor is required for gene expression dynamics in response to extreme oxidative stress in a hypersaline-adapted archaeon

Abstract

BackgroundPrevious work has shown that the hypersaline-adapted archaeon, Halobacterium salinarum NRC-1, is highly resistant to oxidative stress caused by exposure to hydrogen peroxide, UV, and gamma radiation. Dynamic alteration of the gene regulatory network (GRN) has been implicated in such resistance. However, the molecular functions of transcription regulatory proteins involved in this response remain unknown.ResultsHere we have reanalyzed several existing GRN and systems biology datasets for H. salinarum to identify and characterize a novel winged helix-turn-helix transcription factor, VNG0258H, as a regulator required for reactive oxygen species resistance in this organism. This protein appears to be unique to the haloarchaea at the primary sequence level. High throughput quantitative growth assays in a deletion mutant strain implicate VNG0258H in extreme oxidative stress resistance. According to time course gene expression analyses, this transcription factor is required for the appropriate dynamic response of nearly 300 genes to reactive oxygen species damage from paraquat and hydrogen peroxide. These genes are predicted to function in repair of oxidative damage to proteins and DNA. In vivo DNA binding assays demonstrate that VNG0258H binds DNA to mediate gene regulation.ConclusionsTogether these results suggest that VNG0258H is a novel archaeal transcription factor that regulates gene expression to enable adaptation to the extremely oxidative, hypersaline niche of H. salinarum. We have therefore renamed VNG0258H as RosR, for reactive oxygen species regulator.

Detecting differential growth of microbial populations with Gaussian process regression

“…The strain ΔrosR is a known oxidative stress regulator that has previously been shown to regulate oxidative stress under both PQ and hydrogen peroxide exposure (Sharma et al 2012;Tonner et al 2015). Surprisingly, this strain did not exhibit a significant differential growth phenotype versus the parent strain under oxidative stress in our study (Fig.…”

“…In order to determine the source of this discrepancy, we compared the growth data for ΔrosR generated for this study to data from a previous study (Supplemental Fig. S9; Sharma et al 2012). We observed that Δura3 reached a higher cell density in stationary phase than ΔrosR under standard conditions, showing a significant BF score (FDR ≤ 20%) in our study (Fig.…”

“…Chronic oxidative stress was induced by the addition of 0.333 mM paraquat (PQ) when the culture was inoculated. The growth rate of these TF strains under standard conditions during log phase has been tested previously (Kaur et al 2006;Schmid et al 2009Schmid et al , 2011Sharma et al 2012;Plaisier et al 2014), but only the growth rates of TF knockout mutants ΔasnC, ΔtrmB, and ΔrosR have been tested under PQ conditions (Table 1; Schmid et al 2009;Sharma et al 2012;Plaisier et al 2014). …”

“…The fifth strain with a novel differential growth phenotype, ΔasnC, is impaired for growth throughout the time course. Although the growth of Δidr1, Δidr2, ΔrosR, and ΔasnC strains has been studied during log phase under standard growth conditions previously (Schmid et al 2011;Sharma et al 2012;Plaisier et al 2014), these represent novel stationary phase phenotypes. Taken together, these results demonstrate that B-GREAT and the OD Δ metric provide a simple, biologically interpretable test of significance of differential growth that captures the complexity of growth phenotypes.…”

“…A gene regulatory network inferred from transcriptomic data predicts that over 70 transcription factors (TFs) may regulate genes whose products adjust physiology and repair damage incurred by stress (Bonneau et al 2007;Brooks et al 2014). Network predictions have been used to characterize the full set of TF target genes and physiological roles of TFs that control the response to oxidative stress through RosR and AsnC (Sharma et al 2012;Plaisier et al 2014;Tonner et al 2015), nutrient availability through TrmB (Schmid et al 2009;Todor et al 2013Todor et al , 2014, metals through SirR (Kaur et al 2006), iron homeostasis through Idr1 and Idr2 (Schmid et al 2011), and copper response through VNG1179C (Kaur et al 2006;Plaisier et al 2014). Despite this knowledge, the cellular regulators of growth that respond to environmental perturbation remain understudied in H. salinarum and other archaeal species.…”