Extensive functional divergence of the RosR transcription factor across haloarchaea

For microbial cells, an appropriate response to changing environmental conditions is critical for viability. Transcription regulatory proteins, or transcription factors (TFs) sense environmental signals to change gene expression. However, it remains unclear how TFs and their corresponding gene regulatory networks are selected for over evolutionary time scales. The function of TFs and how they evolve are particularly understudied in archaeal organisms. Here we identified, characterized, and compared the function of the RosR transcription factor across three related species of hypersaline adapted model archaea. RosR was previously characterized as a global regulator of gene expression during oxidative stress in the species Halobacterium salinarum (HsRosR). Here we use functional genomics and quantitative phenotyping to demonstrate that, despite strong conservation of HsRosR across species, its function diverges substantially. Surprisingly, RosR in Haloferax volcanii and Haloferax mediterranei regulates genes whose products function in motility and the outer membrane, leading to significant defects in motility when rosR is deleted. Given weak conservation and degeneration in the RosR cis-regulatory sequence across species, we hypothesize that the RosR regulatory network is easily rewired during evolution across related species of archaea. Overall design: To determine the function of the RosR transcription factor across the species Haloferax volcanii (HVO), Haloferax mediterranei (HFX), we measured RNA expression by RNA-seq transcriptomics in rosR knockout strains of each species compared to its corresponding parent strain (ura3 , HBT; pyrE in HVO and HFX)