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).

Keywords: Atacama Desert; DNA repair; Mars; hypersaline environments; oxidative stress; perchlorate-resistance; protein damage; tRNA modification.

FIGURE 1

Drop assays (A) and survival percentage (B) of the nine complete clones and individual predicted ORFs involved in perchlorate-resistance. Cultures were grown overnight at 37°C, drops of 10-fold serial dilutions were plated on LB-Ap containing 125 mM of sodium perchlorate and plates were incubated for 48 h at 37°C. Survival percentage was calculated as the number of CFU mL^–1 remaining after the treatment divided by the initial CFU mL^–1. E. coli DH10B bearing empty pSKII+ (pBlueScript II SK +) vector was used as a negative control. One-way ANOVA followed by Dunnet post hoc analysis revealed the displayed significant differences against the negative control (***p < 0.001). Data represent the mean ± S.D (n ≥ 3).

FIGURE 2

Schematic organization of the predicted ORFs identified in plasmids pJR10 to pJR157. Arrows indicate the locations and the transcriptional orientation of the ORFs in the different plasmids. Those predicted ORFs involved in perchlorate resistance are denoted by blue arrows. Truncated ORFs are indicated by a vertical bar at the corresponding end.

FIGURE 3

Drop assays in the presence of perchlorate of clones overexpressing: (A) pJR12-orf2, E. coli DH10B queF and B. subtilis PY79 queF, and (B) pJR76, pJR76-orf2 and E. coli DH10B yrbC. Cultures were grown overnight at 37°C, drops of 2-fold (A) or 10-fold (B) serial dilutions were plated on LB-Ap containing 125 mM of sodium perchlorate and plates were incubated for 48 h at 37°C. E. coli DH10B bearing empty pSKII+ (pBlueScript II SK +) vector was used as a negative control.

FIGURE 4

UV-B resistance test by drop assays of the individual predicted ORFs involved in perchlorate resistance and their complete clones. Cultures were grown overnight at 37°C, drops of 4-fold serial dilutions were plated on LB-Ap and plates were incubated for 24 h after exposure to 20 s of UV-B (312 nm) radiation at 2 W m^–2.

FIGURE 5

4-NQO (4-nitroquinoline-N-oxide) resistance test by drop assays of the individual predicted ORFs involved in perchlorate resistance and their complete clones. Cultures were grown overnight at 37°C, drops of 4-fold serial dilutions were plated on LB-Ap supplemented with 0.2 μM 4-NQO and plates were incubated for 48 h at 37°C.

FIGURE 6

Hydrogen peroxide resistance test by drop assays of the individual predicted ORFs involved in perchlorate resistance and their complete clones. Cultures were grown overnight at 37°C, drops of 2-fold serial dilutions were plated on LB-Ap supplemented with 1 mM H₂O₂ and plates were incubated for 24 h at 37°C.