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Random mutagenesis of the hyperthermophilic archaeon Pyrococcus furiosus using in vitro mariner transposition and natural transformation.

ABSTRACT: Transposition mutagenesis is a powerful tool to identify the function of genes, reveal essential genes and generally to unravel the genetic basis of living organisms. However, transposon-mediated mutagenesis has only been successfully applied to a limited number of archaeal species and has never been reported in Thermococcales. Here, we report random insertion mutagenesis in the hyperthermophilic archaeon Pyrococcus furiosus. The strategy takes advantage of the natural transformability of derivatives of the P. furiosus COM1 strain and of in vitro Mariner-based transposition. A transposon bearing a genetic marker is randomly transposed in vitro in genomic DNA that is then used for natural transformation of P. furiosus. A small-scale transposition reaction routinely generates several hundred and up to two thousands transformants. Southern analysis and sequencing showed that the obtained mutants contain a single and random genomic insertion. Polyploidy has been reported in Thermococcales and P. furiosus is suspected of being polyploid. Yet, about half of the mutants obtained on the first selection are homozygous for the transposon insertion. Two rounds of isolation on selective medium were sufficient to obtain gene conversion in initially heterozygous mutants. This transposition mutagenesis strategy will greatly facilitate functional exploration of the Thermococcales genomes.

SUBMITTER: Guschinskaya N

PROVIDER: S-EPMC5099854 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Random mutagenesis of the hyperthermophilic archaeon Pyrococcus furiosus using in vitro mariner transposition and natural transformation.

Guschinskaya Natalia N Brunel Romain R Tourte Maxime M Lipscomb Gina L GL Adams Michael W W MWW Oger Philippe P Charpentier Xavier X

Scientific reports 20161108

Transposition mutagenesis is a powerful tool to identify the function of genes, reveal essential genes and generally to unravel the genetic basis of living organisms. However, transposon-mediated mutagenesis has only been successfully applied to a limited number of archaeal species and has never been reported in Thermococcales. Here, we report random insertion mutagenesis in the hyperthermophilic archaeon Pyrococcus furiosus. The strategy takes advantage of the natural transformability of deriva ...[more]

PMID: 27824140

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Project description:Oxidative Stress Protection and the Repair Response To Hydrogen Peroxide in the Hyperthermophilic Archaeon Pyrococcus furiosus Pyrococcus furiosus is a shallow marine, anaerobic archaeon that grows optimally at 100Â°C. Addition of H2O2 (0.5 mM) to a growing culture resulted in cessation of growth with a 2 hour lag before normal growth resumed. Whole genome transcriptional profiling revealed that the main response occurs within 30 min of peroxide addition, with the up-regulation of 62 open reading frames (ORFs), 36 of which are part of 10 potential operons. More than half of the up-regulated ORFs are of unknown function while some others encode proteins that are involved potentially in sequestering iron and sulfide, in DNA repair and in generating NADPH. This response is thought to involve primarily damage repair rather than protection, since cultures exposed to sub-toxic levels of H2O2 were not more resistant to the subsequent addition of H2O2 (0.5 â 5.0 mM). Consequently, there is little if any induced protective response to peroxide, rather, the organism maintains a constitutive protective mechanism involving high levels of oxidoreductase-type enzymes such as superoxide reductase, rubrerythrin and alkyl hydroperoxide reductase I. The related hyperthermophiles P. woesei and Thermococcus kodakaraensis were more sensitive to peroxide than P. furiosus, apparently due to the lack of several of its peroxide-responsive ORFs. Pyrococcus furiosus (DSM 3638) was grown at 95Â°C in a 20-liter fermentor using maltose as the carbon and energy source. An exponential-phase culture of P. furiosus that had undergone three successive transfers in the experimental medium was used to inoculate the 20-liter fermentor. The culture was shocked with 0.5 mM of hydrogen peroxide (H2O2) when cell density was in mid-exponential phase (~ 5.0 Â´ 107 cells/ml, as determined by direct microscopic cell count). To obtain RNA for microarray and for quantitative PCR (QPCR) analyses, samples (2 liter) were rapidly removed from the fermentor and cooled to 4Â°C. Total RNA was extracted using acid-phenol and stored at -80Â°C until needed. A total of 3 biological replicates in triplicate (3 copies on the same slide) was used in the data set.

Project description:Insights into the Metabolism of Elemental Sulfur by the Hyperthermophilic Archaeon Pyrococcus furiosus: Characterization of a Coenzyme A-Dependent NAD(P)H Sulfur Oxidoreductase The hyperthermophilic archaeon Pyrococcus furiosus, uses carbohydrates as a carbon source and produces acetate, CO2 and H2 as end products. When S° is added to a growing culture, within 10 min the rate of H2 production rapidly decreases and H2S is detected. After one hour cells contain high NADPH- and coenzyme A-dependent S° reduction activity (0.7 units/mg, 85°C) located in the cytoplasm. The enzyme responsible for this activity was purified to electrophoretic homogeneity (specific activity, 100 units/mg) and is termed NAD(P)H elemental sulfur oxidoreductase (NSR). NSR is a homodimeric flavoprotein (Mr 100 kDa) and is encoded by PF1186. This was previously assigned to an enzyme that reduces coenzyme A disulfide, which is a side-reaction of NSR. Whole genome DNA microarray and quantitative PCR analyses showed that the expression of NSR is up-regulated up to 7-fold within 10 min of S° addition. This primary response to S° also involves the up-regulation (> 16-fold) of a 13 gene cluster encoding a membrane-bound oxidoreductase (MBX). MBX is proposed replace the homologous 14 gene cluster that encodes the ferredoxin-oxidizing, H2-evolving membrane-bound hydrogenase (MBH), which is down-regulated >12-fold within 10 min of S° addition. Although an activity for MBX could not be demonstrated, it is proposed to conserve energy by oxidizing ferredoxin and reducing NADP, which is used by NSR to reduce S°. A secondary response to S° is observed 30 min after S° addition and includes the up-regulation of genes encoding proteins involved in amino acid biosynthesis and iron metabolism, as well as two so-called sulfur-induced proteins, termed SipA and SipB. This novel S°-reducing system involving NSR and MBX is so far unique to the heterotrophic Thermococcales, and is in contrast to the cytochrome- and quinone-based S°-reducing system in autotrophic archaea and bacteria. Keywords: time course, kinetic, sulfur metabolism, archaea, Pyrococcus furiosus, hyperthermophile

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Random mutagenesis of the hyperthermophilic archaeon Pyrococcus furiosus using in vitro mariner transposition and natural transformation.

Publications

Random mutagenesis of the hyperthermophilic archaeon Pyrococcus furiosus using in vitro mariner transposition and natural transformation.

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