TbsP and TrmB jointly regulate gapII to influence cell development phenotypes in the archaeon Haloferax volcanii.

Microbial cells must continually adapt their physiology in the face of changing environmental conditions. Archaea living in extreme conditions, such as saturated salinity, represent important examples of such resilience. The model salt-loving organism Haloferax volcanii exhibits remarkable plasticity in its morphology, biofilm formation, and motility in response to variations in nutrients and cell density. However, the mechanisms regulating these lifestyle transitions remain unclear. In prior research, we showed that the transcriptional regulator, TrmB, maintains the rod shape in the related species Halobacterium salinarum by activating the expression of enzyme-coding genes in the gluconeogenesis metabolic pathway. In Hbt. salinarum, TrmB-dependent production of glucose moieties is required for cell surface glycoprotein biogenesis. Here, we use a combination of genetics and quantitative phenotyping assays to demonstrate that TrmB is essential for growth under gluconeogenic conditions in Hfx. volcanii. The ∆trmB strain rapidly accumulated suppressor mutations in a gene encoding a novel transcriptional regulator, which we name trmB suppressor, or TbsP (a.k.a. "tablespoon"). TbsP is required for adhesion to abiotic surfaces (i.e., biofilm formation) and maintains wild-type cell morphology and motility. We use functional genomics and promoter fusion assays to characterize the regulons controlled by each of TrmB and TbsP, including joint regulation of the glucose-dependent transcription of gapII, which encodes an important gluconeogenic enzyme. We conclude that TrmB and TbsP coregulate gluconeogenesis, with downstream impacts on lifestyle transitions in response to nutrients in Hfx. volcanii.

Comparative Analysis of rRNA Removal Methods for RNA-Seq Differential Expression in Halophilic Archaea.

Despite intense recent research interest in archaea, the scientific community has experienced a bottleneck in the study of genome-scale gene expression experiments by RNA-seq due to the lack of commercial and specifically designed rRNA depletion kits. The high rRNA:mRNA ratio (80-90%: ~10%) in prokaryotes hampers global transcriptomic analysis. Insufficient ribodepletion results in low sequence coverage of mRNA, and therefore, requires a substantially higher number of replicate samples and/or sequencing reads to achieve statistically reliable conclusions regarding the significance of differential gene expression between case and control samples. Here, we show that after the discontinuation of the previous version of RiboZero (Illumina, San Diego, CA, USA) that was useful in partially or completely depleting rRNA from archaea, archaeal transcriptomics studies have experienced a slowdown. To overcome this limitation, here, we analyze the efficiency for four different hybridization-based kits from three different commercial suppliers, each with two sets of sequence-specific probes to remove rRNA from four different species of halophilic archaea. We conclude that the key for transcriptomic success with the currently available tools is the probe-specificity for the rRNA sequence hybridization. With this paper, we provide insights into the archaeal community for selecting certain reagents and strategies over others depending on the archaeal species of interest. These methods yield improved RNA-seq sensitivity and enhanced detection of low abundance transcripts.

Mitochondrial function is an inducible determinant of osmotic stress adaptation in yeast.

Hyperosmotic stress triggers a great variety of adaptive responses in eukaryotic cells that affect many different physiological functions. Here we investigate the role of the mitochondria during osmostress adaptation in budding yeast. Mitochondrial function is generally required for proper salt and osmotic stress adaptation because mutants with defects in many different mitochondrial components show hypersensitivity to increased NaCl and KCl concentrations. Mitochondrial protein abundance rapidly increases upon osmoshock in a selective manner, because it affects Calvin cycle enzymes (Sdh2 and Cit1) and components of the electron transport chain (Cox6) but not the ATP synthase complex (Atp5). Transcription of the SDH2, CIT1, and COX6 genes is severalfold induced within the first minutes of osmotic shock, dependent to various degree on the Hog1 and Snf1 protein kinases. Mitochondrial succinate dehydrogenase enzyme activity is stimulated upon osmostress in a Snf1-dependent manner. The osmosensitivity of mitochondrial mutants is not caused by impaired stress-activated transcription or by a general depletion of the cellular ATP pool during osmostress. We finally show that the growth defect of mitochondrial mutants in high salt medium can be partially rescued by supplementation of glutathione. Additionally, mitochondrial defects cause the hyperaccumulation of reactive oxygen species during salt stress. Our results indicate that the antioxidant function of the mitochondria might play an important role in adaptation to hyperosmotic stress.