Discovery of novel hyperthermophilic DNA modifying enzymes using a functional genomics screen
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ABSTRACT:
ORGANISM(S): Thermococcus kodakarensis
PROVIDER: GSE182067 | GEO | 2021/08/18
REPOSITORIES: GEO
Similar Datasets
Project description:Discovery of novel hyperthermophilic DNA modifying enzymes using a functional genomics screen
| PRJNA754396 | ENA
Project description:A hyperthermophilic archaeon Thermococcus kodakarensis can grow in a wide range of temperatures from 60°C to 93°C, optimally at 85°C. To screen the genes that being specifically expressed at lowest growth temperature, 60°C, we investigated the transcriptional profilings of T. kodakarensis grown at 60˚C by comparing with those grown at 85°C.
2016-04-01 | GSE71987 | GEO
Project description:A hyperthermophilic archaeon Thermococcus kodakarensis can grow in a wide range of temperatures from 60°C to 93°C, optimally at 85°C. To screen the genes that being specifically expressed at lowest growth temperature, 60°C, we investigated the transcriptional profilings of T. kodakarensis grown at 60ËC by comparing with those grown at 85°C. One-condition experiment, KU216 grown at optimal growth temperature, 85°C vs. KU216 grown at lowest growth temperature, 60°C. Technical replicates: 2 KU216 grown at 60°C, independently measured. One replicate per array.
2016-04-01 | E-GEOD-71987 | biostudies-arrayexpress
Project description:Thermococcus kodakarensis preferentially utilizes amino acids as carbon and energy sources in the presence of elemental sulfur as a terminal electron acceptor, while it can assimilate and grow on starch or pyruvate using proton as a terminal acceptor, generating hydrogen in the absence of elemental sulfur. SurR is a transcriptional regulator controlling hydrogen and elemental sulfur metabolism. To identify the genes that are under the regulation of Tk-SurR, we investigated the transcriptional profiling of Tk-SurR deletion strain grown in the presence of elemental sulfur at 85˚C by comparing with the host strain, KU216.
2016-01-01 | GSE71984 | GEO
Project description:Thermococcus kodakarensis preferentially utilizes amino acids as carbon and energy sources in the presence of elemental sulfur as a terminal electron acceptor, while it can assimilate and grow on starch or pyruvate using proton as a terminal acceptor, generating hydrogen in the absence of elemental sulfur. SurR is a transcriptional regulator controlling hydrogen and elemental sulfur metabolism. To identify the genes that are under the regulation of Tk-SurR, we investigated the transcriptional profiling of Tk-SurR deletion strain grown in the presence of elemental sulfur at 85ËC by comparing with the host strain, KU216. One-condition experiment, KU216 vs. DTS cells. Technical replicates: 2 DTS grown in the presence of elemental sulfur at 85ËC, independently measured. One replicate per array.
2016-07-03 | E-GEOD-71984 | biostudies-arrayexpress
Project description:Thermococcus kodakarensis, which can grow in a wide range of temperatures, from 60°C to 100°C, optimally at 85°C, has two transcription factor Bs, TFB1 and TFB2, both of which are basal transcription factor. TFB1 is unique to hyperthermophiles, but TFB2 is widely conserved in Euryarchaeota. To screen the genes dependent on each TFB in a temperature dependent manner, we investigated the transcriptional profilings of each TFB deletant strain grown at each temperature, 60˚C, 85˚C, and 93˚C, by comparing with the host strain, KU216. Data analyses showed that TFB1 apparently controls the expression of genes essential for motility and adhesion, such as flagellin genes, at 85˚C and 93˚C, but TFB2 regulates genes involved in basal metabolism including amino acid biosynthesis.
2013-08-14 | GSE49841 | GEO
Project description:Background: Prokaryotes have relatively small genomes, densely-packed and apparently dominated by protein-encoding sequences. However, data now generated by high throughput RNA sequencing (RNA-seq) reveal surprisingly more-complex transcriptomes with many previously unrecognized and unanticipated non-coding small and antisense transcripts. To date, such studies have investigated primarily Bacteria. Here, we report the transcripts present in Thermococcus kodakarensis, a model hyperthermophilic Archaeon, synthesized under different growth and metabolic conditions. Results: cDNA libraries, generated from RNA preparations isolated from cells growing in media with sulfur or pyruvate, with sulfur to stationary phase, and growing with pyruvate but with sulfur added 20 min before RNA isolation, have been deep-sequenced. The results identify >2,700 sites of transcription initiation, establish a genome-wide map of transcripts, and consensus sequences for transcription initiation and post-transcription regulatory elements in T. kodakarensis. Primary transcription start sites (TSS) are identified upstream of 1,254 annotated genes, including ~78 % of those predicted by promoter locations, and an additional 644 primary TSS and their promoters have been identified within genes. Most of the mRNAs have a 5'-untranslated region (5'-UTR) between 10 and 50 nt long (median length = 16 nt), ~20 % have 5'-UTRs from 50 to 300 nt long, ~14 % are leaderless with 5'-UTRs ≤8 nt, and ~50% contain a consensus ribosome binding sequence. The results also identify TSS for 1,018 antisense transcripts, most with sequences complementary to either the 5'- or 3'-region of a sense mRNA. The data confirm the presence of transcripts from all three CRISPR loci, the RNase P and 7S RNAs, all tRNAs and rRNAs and 69 snoRNAs predicted to be encoded in the T. kodakarensis genome. Two transcripts, putatively identified as riboswitches, were present in RNA preparations isolated from growing but not from stationary phase cells. The procedure used is designed to identify TSS but, assuming that the number of cDNA reads correlates with transcript abundance, the data obtained also provide a semi-quantitative overview of global operon expression. They document substantial differences in gene expression under different physiological conditions and are consistent with previous observations of substrate-dependent specific gene expression. Many previously unrecognized and unanticipated small RNAs have been identified, some with relative low GC contents (≤50%) and sequences that do not fold readily into base-paired secondary structures, contrary to the classical expectations for non-coding RNAs in a hyperthermophile. Conclusion: We have identified >2,700 TSS that include almost all of the primary sites of transcription initiation upstream of annotated genes, and also many secondary sites, sites within genes and sites resulting in antisense transcripts. The T. kodakarensis genome is small (~2.1 Mbp) and tightly packed with protein-encoding genes, but the results reveal the presence of many non-coding RNAs and predict extensive RNA-based regulation in T. kodakarensis.
2014-08-18 | GSE56262 | GEO
Project description:Thermococcus kodakarensis KOD1 Epigenomics
| PRJNA209412 | ENA
Project description:It has been predicted that 30 to 80% of archaeal genomes remain annotated as hypothetical proteins with no assigned gene function. Further, many archaeal organisms are difficult to grow or are unculturable. To overcome these technical and experimental hurdles, we developed a high-throughput functional genomics screen that utilizes capillary electrophoresis (CE) to identify nucleic acid modifying enzymes based on activity rather than sequence homology. Here, we describe a functional genomics screening workflow to find DNA modifying enzyme activities encoded by the hyperthermophile Thermococcus kodakarensis (T. kodakarensis). Large DNA insert fosmid libraries representing an ∼5-fold average coverage of the T. kodakarensis genome were prepared in Escherichia coli. RNA-seq showed a high fraction (84%) of T. kodakarensis genes were transcribed in E. coli despite differences in promoter structure and translational machinery. Our high-throughput screening workflow used fluorescently labeled DNA substrates directly in heat-treated lysates of fosmid clones with capillary electrophoresis detection of reaction products. Using this method, we identified both a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and a novel AP lyase DNA repair enzyme family (termed 'TK0353') that is found only in a small subset of Thermococcales. The screening methodology described provides a fast and efficient way to explore the T. kodakarensis genome for a variety of nucleic acid modifying activities and may have implications for similar exploration of enzymes and pathways that underlie core cellular processes in other Archaea. IMPORTANCE This study provides a rapid, simple, high-throughput method to discover novel archaeal nucleic acid modifying enzymes by utilizing a fosmid genomic library, next-generation sequencing, and capillary electrophoresis. The method described here provides the details necessary to create 384-well fosmid library plates from Thermococcus kodakarensis genomic DNA, sequence 384-well fosmids plates using Illumina next-generation sequencing, and perform high-throughput functional read-out assays using capillary electrophoresis to identify a variety of nucleic acid modifying activities, including DNA cleavage and ligation. We used this approach to identify a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and identify a novel AP lyase enzyme (TK0353) that lacks sequence homology to known nucleic acid modifying enzymes.
| S-EPMC8788744 | biostudies-literature
Project description:Thermococcus kodakarensis bisulfite-sequenced RNA
| PRJNA937301 | ENA