Project description:Antisense RNAs (asRNAs) are present in diverse organisms and play important roles in gene regulation. In this work, we mapped the primary antisense transcriptome in the halophilic archaeon Halobacterium salinarum NRC-1. By reanalyzing publicly available data, we mapped antisense transcription start sites (aTSSs) and inferred the probable 3' ends of these transcripts. We analyzed the resulting asRNAs according to the size, location, function of genes on the opposite strand, expression levels and conservation. We show that at least 21% of the genes contain asRNAs in H. salinarum. Most of these asRNAs are expressed at low levels. They are located antisense to genes related to distinctive characteristics of H. salinarum, such as bacteriorhodopsin, gas vesicles, transposases and other important biological processes such as translation. We provide evidence to support asRNAs in type II toxin⁻antitoxin systems in archaea. We also analyzed public Ribosome profiling (Ribo-seq) data and found that ~10% of the asRNAs are ribosome-associated non-coding RNAs (rancRNAs), with asRNAs from transposases overrepresented. Using a comparative transcriptomics approach, we found that ~19% of the asRNAs annotated in H. salinarum belong to genes with an ortholog in Haloferax volcanii, in which an aTSS could be identified with positional equivalence. This shows that most asRNAs are not conserved between these halophilic archaea.
Project description:RosR is a haloarchaeal-specific transcription factor required for the response to extreme oxidative stress in Halobacterium salinarum NRC-1.
Project description:Previous 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. Genome-wide dynamics alteration of gene the GRN has been implicated in such resistance. However, the molecular function of transcription regulatory proteins involved in this response remains unknown. Here we have leveraged 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. 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 (ChIP-qPCR) demonstrate that VNG0258H binds DNA to mediate gene regulation. Together these results suggest that VNG0258H is a novel archaeal transcription regulatory protein 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. Data in this archive are linked to the publication Sharma KS, Gillum NA, Schmid AK 2012
Project description:Halobacterium salinarum NRC-1 was grown in CM media, at 37oC in a waterbath with agitation of 125 rpm under constant light. Analysis of transcriptional changes during growth, in addition to mapping of transcriptome structure under the same conditions, provided interesting insights about regulatory logic within prokaryotic coding regions.
Project description:Gene regulatory networks play an important role in coordinating biochemical fluxes through diverse metabolic pathways. The modulation of enzyme levels enables efficient utilization of limited resources as organisms dynamically acclimate to nutritional fluctuations in their environment. Here we have identified and characterized a novel nutrient-responsive transcription factor from the halophilic archaea, AgmR. Like TrmB, its thermophilic archaeal homolog, AgmR regulates glycolytic and gluconeogenic pathways in response to sugar availability. However, using high throughput genome-scale experiments, we find that AgmR directly governs the transcription of nearly 100 additional genes encoding enzymes in diverse metabolic pathways. Genome-scale in vivo binding site location data reveals that >60% of these are direct targets. Integration of these systems-scale datasets with metabolic reconstruction models suggests that AgmR, a sequence-specific bacterial-like regulator, interacts with the general transcription factor machinery to coordinate nitrogen and carbon metabolism with the de novo synthesis of cognate cofactors and reducing equivalents, achieving system-wide redox and energy balance.
Project description:Previous 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. Genome-wide dynamics alteration of gene the GRN has been implicated in such resistance. However, the molecular function of transcription regulatory proteins involved in this response remains unknown. Here we have leveraged 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. 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 (ChIP-qPCR) demonstrate that VNG0258H binds DNA to mediate gene regulation. Together these results suggest that VNG0258H is a novel archaeal transcription regulatory protein 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. Data in this archive are linked to the publication Sharma KS, Gillum NA, Schmid AK 2012 The M-NM-^Tura3 and M-NM-^TrosR strains were grown to mid-logarithmic phase (OD600 ~ 0.5) in CM supplemented with uracil. For the H2O2 time courses, 4 ML culture aliquots were removed for RNA extraction at three time points prior to the addition of H2O2 (-40 min, -20 min, 0min) and five time points following H2O2 addition (10 min, 20 min, 40 min, 60 min, 80 min). Paraquat time courses were prepared similarly with the exception that additional time points were taken at 2h, 8h, and 24 h after the addition of paraquat. RNA from two biological replicate time courses were prepared, along with a dye filip.
Project description:Sustained bioenergy production from organisms that thrive in high salinity, low oxygen, and low nutrition levels is useful in monitoring hypersaline polluted environments. Microbial fuel cell (MFC) studies utilizing single species halophiles under salt concentrations higher than 1 M and as a closed microbial system are limited. The current study aimed to establish baseline voltage, current, and power density from a dual-chambered MFC utilizing the halophile Halobacterium salinarum NRC-1. MFC performance was determined with two different electrode sizes (5 cm2 and 10 cm2), under oscillating and nonoscillating conditions, as well as in a stacked series. A closed dual-chamber MFC system of 100 mL capacity was devised with Halobacterium media (4.3 M salt concentration) as both anolyte and catholyte, with H. salinarum NRC-1 being the anodic organism. The MFC measured electrical output over 7, 14, 28, and 42 days. MFC output increased with 5 cm2 sized electrodes under nonoscillating (p < 0.0001) relative to oscillating conditions. However, under oscillating conditions, doubling the electrode size increased MFC output significantly (p = 0.01). The stacked series MFC, with an electrode size of 10 cm2, produced the highest power density (1.2672 mW/m2) over 14 days under oscillation. Our results highlight the potentiality of H. salinarum as a viable anodic organism to produce sustained voltage in a closed-MFC system.