Mining for Perchlorate Resistance Genes in Microorganisms From Sediments of a Hypersaline Pond in Atacama Desert, Chile.

Perchlorate is an oxidative pollutant toxic to most of terrestrial life by promoting denaturation of macromolecules, oxidative stress, and DNA damage. However, several microorganisms, especially hyperhalophiles, are able to tolerate high levels of this compound. Furthermore, relatively high quantities of perchlorate salts were detected on the Martian surface, and due to its strong hygroscopicity and its ability to substantially decrease the freezing point of water, perchlorate is thought to increase the availability of liquid brine water in hyper-arid and cold environments, such as the Martian regolith. Therefore, perchlorate has been proposed as a compound worth studying to better understanding the habitability of the Martian surface. In the present work, to study the molecular mechanisms of perchlorate resistance, a functional metagenomic approach was used, and for that, a small-insert library was constructed with DNA isolated from microorganisms exposed to perchlorate in sediments of a hypersaline pond in the Atacama Desert, Chile (Salar de Maricunga), one of the regions with the highest levels of perchlorate on Earth. The metagenomic library was hosted in Escherichia coli DH10B strain and exposed to sodium perchlorate. This technique allowed the identification of nine perchlorate-resistant clones and their environmental DNA fragments were sequenced. A total of seventeen ORFs were predicted, individually cloned, and nine of them increased perchlorate resistance when expressed in E. coli DH10B cells. These genes encoded hypothetical conserved proteins of unknown functions and proteins similar to other not previously reported to be involved in perchlorate resistance that were related to different cellular processes such as RNA processing, tRNA modification, DNA protection and repair, metabolism, and protein degradation. Furthermore, these genes also conferred resistance to UV-radiation, 4-nitroquinoline-N-oxide (4-NQO) and/or hydrogen peroxide (H2O2), other stress conditions that induce oxidative stress, and damage in proteins and nucleic acids. Therefore, the novel genes identified will help us to better understand the molecular strategies of microorganisms to survive in the presence of perchlorate and may be used in Mars exploration for creating perchlorate-resistance strains interesting for developing Bioregenerative Life Support Systems (BLSS) based on in situ resource utilization (ISRU).

Elongator Subunit 3 (Elp3) Is Required for Zebrafish Trunk Development.

Transfer RNAs (tRNAs) are the most post-transcriptionally modified RNA species. Some of these modifications, especially the ones located in the anti-codon loop, are required for decoding capabilities of tRNAs. Such is the case for 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U), synthetized by the Elongator complex. Mutants for its sub-units display pleiotropic phenotypes. In this paper, we analyze the role of elp3 (Elongator catalytic sub-unit) in zebrafish development. We found that it is required for trunk development; elp3 knock-down animals presented diminished levels of mcm5s2U and sonic hedgehog (Shh) signaling activity. Activation of this pathway was sufficient to revert the phenotype caused by elp3 knockdown, indicating a functional relationship between Elongator and Shh through a yet unknown molecular mechanism.

KAE1 Allelic Variants Affect TORC1 Activation and Fermentation Kinetics in Saccharomyces cerevisiae.

The eukaryotic domain-conserved TORC1 signalling pathway connects growth with nutrient sufficiency, promoting anabolic processes such as ribosomal biogenesis and protein synthesis. In Saccharomyces cerevisiae, TORC1 is activated mainly by the nitrogen sources. Recently, this pathway has gotten renewed attention but now in the context of the alcoholic fermentation, due to its key role in nitrogen metabolism regulation. Although the distal and proximal effectors downstream TORC1 are well characterised in yeast, the mechanism by which TORC1 is activated by nitrogen sources is not fully understood. In this work, we took advantage of a previously developed microculture-based methodology, which indirectly evaluates TORC1 activation in a nitrogen upshift experiment, to identify genetic variants affecting the activation of this pathway. We used this method to phenotype a recombinant population derived from two strains (SA and WE) with different geographic origins, which show opposite phenotypes for TORC1 activation by glutamine. Using this phenotypic information, we performed a QTL mapping that allowed us to identify several QTLs for TORC1 activation. Using a reciprocal hemizygous analysis, we validated GUS1, KAE1, PIB2, and UTH1 as genes responsible for the natural variation in the TORC1 activation. We observed that reciprocal hemizygous strains for KAE1 (ATPase required for t6A tRNA modification) gene showed the greatest phenotypic differences for TORC1 activation, with the hemizygous strain carrying the SA allele (KAE1 SA ) showing the higher TORC1 activation. In addition, we evaluated the fermentative capacities of the hemizygous strains under low nitrogen conditions, observing an antagonistic effect for KAE1 SA allele, where the hemizygous strain containing this allele presented the lower fermentation rate. Altogether, these results highlight the importance of the tRNA processing in TORC1 activation and connects this pathway with the yeasts fermentation kinetics under nitrogen-limited conditions.

Protein Phosphatase Sit4 Affects Lipid Droplet Synthesis and Soraphen A Resistance Independent of Its Role in Regulating Elongator Dependent tRNA Modification.

The protein phosphatase Sit4 has been shown to be required for lipogenesis and resistance against the acetyl-CoA carboxylase inhibitor soraphen A. Since Sit4 is also required for biosynthesis of Elongator dependent tRNA modifications such as 5-methoxycarbonylmethyluridine (mcm5U), we investigated the relevance of tRNA modifications in lipogenesis and soraphen A response. While sit4 and Elongator (elp3) mutants copy defects in mcm5U formation and stress sensitivity, they do not share soraphen A sensitivity and low lipid droplet (LD) phenotypes. In contrast to sit4, we found elp3 mutants to display partial soraphen A resistance and a high LD phenotype. Screening a collection of tRNA modification mutants additionally identified the tRNA pseudo-uridine synthase gene DEG1 to be required for soraphen A sensitivity. Since deg1 and elp3 share high LD and soraphen A resistance phenotypes, these are likely caused by translational defects. In support of this notion, we observe overexpression of tRNAGlnUUG suppresses lipolysis defects of deg1 mutants. Hence, the sit4 mutation results in a composite defect including tRNA modification deficiency and loss of Snf1 kinase dephosphorylation, which induce opposite effects on LD regulation. Importantly, however, the Snf1 kinase regulatory defects of the phosphatase mutant dominate over effects on LD regulation imposed by loss of the tRNA modification alone.

The Drosophila EKC/KEOPS complex: roles in protein synthesis homeostasis and animal growth.

The TOR signaling pathway is crucial in the translation of nutritional inputs into the protein synthesis machinery regulation, allowing animal growth. We recently identified the Bud32 (yeast)/PRPK (human) ortholog in Drosophila, Prpk (p53-related protein kinase), and found that it is required for TOR kinase activity. Bud32/PRPK is an ancient and atypical kinase conserved in evolution from Archeae to humans, being essential for Archeae. It has been linked with p53 stabilization in human cell culture and its absence in yeast causes a slow-growth phenotype. This protein has been associated to KEOPS (kinase, putative endopeptidase and other proteins of small size) complex together with Kae1p (ATPase), Cgi-121 and Pcc1p. This complex has been implicated in telomere maintenance, transcriptional regulation, bud site selection and chemical modification of tRNAs (tRNAs). Bud32p and Kae1p have been related with N6-threonylcarbamoyladenosine (t (6)A) synthesis, a particular chemical modification that occurs at position 37 of tRNAs that pair A-starting codons, required for proper translation in most species. Lack of this modification causes mistranslations and open reading frame shifts in yeast. The core constituents of the KEOPS complex are present in Drosophila, but their physical interaction has not been reported yet. Here, we present a review of the findings regarding the function of this complex in different organisms and new evidence that extends our recent observations of Prpk function in animal growth showing that depletion of Kae1 or Prpk, in accordance with their role in translation in yeast, is able to induce the unfolded protein response (UPR) in Drosophila. We suggest that EKC/KEOPS complex could be integrating t (6)A-modified tRNA availability with translational rates, which are ultimately reflected in animal growth.