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

Abstract

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).

Perchlorate-Specific Proteomic Stress Responses of Debaryomyces hansenii Could Enable Microbial Survival in Martian Brines

“…Oxygen and food production by phototrophic microorganisms and the recycling of waste material in perchlorate-rich Martian soil might be conducted by “chaotolerant” (66) organisms, because they would likely possess a metabolic toolset for stabilization of biomacromolecules similar to D. hansenii . Alternatively, genes responsible for increased biomacromolecular stability might be used in synthetic biology to create perchlorate resistance strains that can thrive in perchlorate-rich Martian soil without the necessity for perchlorate remediation (55).…”

“…Due to the high oxidation state (+7) of the chlorine atom in the center of the perchlorate anion, it is expected that perchlorate exhibits a stronger oxidation stress response than observed in the general salt stress response. Indeed, there is evidence that several genes in microorganisms from 13 sediments of hypersaline ponds increase both the resistance to perchlorate and to oxidative stress induced by hydrogen peroxide (55). Furthermore, increased levels of lipid peroxidation after growth of different species of cyanobacteria in perchlorate-containing growth media were interpreted as results of oxidative stress (56).…”

The smallest space miners: principles of space biomining

“…Microorganisms performing biomining could also be selected for bioremediation approaches to ameliorate these components by making them less toxic or removing them. A recent study identified the genes involved in perchlorate resistance from microorganisms isolated from the Atacama desert (Díaz-Rullo et al 2021 ). A Chroococcidiopsis species and its derivate (CCMEE 029 and CCMEE 029 P-MRS, respectively) had no negative effect to perchlorate exposure, and the biomass produced could be used to feed an heterotrophic bacterial species ( E. coli ; Billi et al 2021 ).…”

Heterologous Expression of the Phytochelatin Synthase CaPCS2 from Chlamydomonas acidophila and Its Effect on Different Stress Factors in Escherichia coli

“…This compound is a potent oxidizing agent that induces bactericidal effects and can be found naturally in extreme arid environments such as the Atacama Desert [103]. Perchlorate is widespread in the global environment [104]. It is a ubiquitous contaminant in water, soil, and food and is toxic to most known microorganisms and humans [105].…”

“…It is a contaminant resulting from anthropogenic activity, and is used in aerospace products, as well as industrial and military applications, such as fertilizers, explosives, fireworks, and rocket fuels [106,107]. Some authors have observed that resistance to UVB is related to resistance to perchlorate exposure, perhaps due to the fact that in both cases there is a high increase in ROS produced by both abiotic stress factors [104,108]. A possible role of PCs in protection against oxidative stress may explain the role of CaPCS2 in tolerance to other abiotic stresses different to metal(loid)s. However, reports on the functions of PCs other than metal chelation are scarce, making it very difficult to draw conclusions.…”